190
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

PhD Thesis Alessandro Carmona2012

Embed Size (px)

Citation preview

Page 1: PhD Thesis Alessandro Carmona2012

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

Page 2: PhD Thesis Alessandro Carmona2012

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

Page 3: PhD Thesis Alessandro Carmona2012

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

Page 4: PhD Thesis Alessandro Carmona2012

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.

Page 5: PhD Thesis Alessandro Carmona2012

„Gedruckt mit Unterstützung des Deutschen Akademischen

Austauschdienstes“

Page 6: PhD Thesis Alessandro Carmona2012

To Yolanda, Jesús and Virginia,

for their love and support...

Page 7: PhD Thesis Alessandro Carmona2012

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.

Page 8: PhD Thesis Alessandro Carmona2012

-i-

Table of contents (brief)

Chapter 1 Extracellular electron transfer in Bioelectrochemical systems: bridge between

natural environments and applied technologies...................................................1

Part I Electron transfer mechanisms of pure culture biofilms of

Shewanella spp.

Chapter 2 Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis

MR-1 and nanofilament and cytochrome knock-out mutants...........................33

Chapter 3 Study of Shewanella putrefaciens biofilms grown at different applied potentials

using cyclic voltammetry and confocal laser scanning microscopy..................47

Chapter 4 Spectroelectrochemical analysis of intact microbial biofilms of Shewanella

putrefaciens for sustainable energy production.................................................61

Part II Porous 3D carbon as anode materials for performance of

electrochemically active mixed culture biofilms

Chapter 5 Electrospun and solution blown three-dimensional carbon fiber nonwovens for

application as electrodes in microbial fuel cells................................................71

Chapter 6 Electrospun carbon fiber mat with layered architecture for anode in microbial

fuel cells.............................................……………………………....................82

Part III The influence of external factors on electrochemically active

mixed culture biofilms

Chapter 7 Electroactive mixed culture derived biofilms in microbial bioelectrochemical

systems: the role of pH on biofilm formation, performance and

composition.......................................................................................................90

Chapter 8 Electroactive mixed culture derived biofilms in microbial bioelectrochemical

systems: the role of inoculum and substrate on biofilm formation and

performance.....................................................................................................108

Page 9: PhD Thesis Alessandro Carmona2012

-ii-

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

Page 10: PhD Thesis Alessandro Carmona2012

-iii-

2 Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis MR-1

and nanofilament and cytochrome knock-out mutants ...................................................... 33

2.1.1.1 Direct electron transfer (DET) .................................................................................................34

2.1.1.2 Mediated electron transfer (MET) ...........................................................................................36

3 Study of Shewanella putrefaciens biofilms grown at different applied potentials

using cyclic voltammetry and confocal laser scanning microscopy.. ................................. 47

2.1 Introduction ..........................................................................................................................................33

2.1.1 Extracellular electron transfer mechanisms of S. oneidensis MR-1 wild type and mutants .............34

2.2 Materials and methods ..........................................................................................................................36

2.2.1 General conditions ...........................................................................................................................36

2.2.2 Cell cultures and media ....................................................................................................................36

2.2.3 Bioelectrochemical experiments ......................................................................................................37

2.2.4 Data processing ................................................................................................................................37

2.3 Results and discussion ..........................................................................................................................38

2.3.1 Bioelectrochemical current production ............................................................................................38

2.3.2 Cyclic voltammetric analysis and data processing ...........................................................................39

2.4 Conclusions ..........................................................................................................................................46

3.1 Introduction ..........................................................................................................................................47

3.1.1 Influence of the electrode potential on electroactive microbial biofilms .........................................49

3.2 Materials and methods ..........................................................................................................................50

3.2.1 General conditions ...........................................................................................................................50

3.2.2 Cell cultures and media ....................................................................................................................50

3.2.3 Bioelectrochemical set-up and experiments .....................................................................................51

3.2.4 Electrochemical data processing ......................................................................................................51

3.2.5 Confocal Laser Scanning Microscopy..............................................................................................52

3.3 Results and discussion ..........................................................................................................................52

3.3.1 Bioelectrochemical current production ............................................................................................52

3.3.2 Cyclic voltammetric analysis ...........................................................................................................54

3.3.3 Biofilm imaging using confocal laser scanning microscopy (CLSM) .............................................58

3.4 Conclusions ..........................................................................................................................................60

Page 11: PhD Thesis Alessandro Carmona2012

-iv-

4 Spectroelectrochemical analysis of intact microbial biofilms of Shewanella

putrefaciens for sustainable energy production ................................................................... 61

5 Electrospun and solution blown three-dimensional carbon fiber nonwovens for

application as electrodes in microbial fuel cells ................................................................... 71

5.2.1.1 Gas-assisted electrospinning carbon fiber mat (GES-CFM) ...................................................73

5.2.1.2 Electrospun carbon fiber mat (ES-CFM) .................................................................................74

5.2.1.3 Solution-blown carbon fiber mat (SB-CFM) ...........................................................................74

4.1 Introduction ..........................................................................................................................................61

4.2 Materials and methods ..........................................................................................................................64

4.2.1 Materials and methods .....................................................................................................................64

4.2.2 Cell cultures and media ....................................................................................................................64

4.2.3 Electrochemical set-up for the growth of anodic electrocatalytic biofilms ......................................65

4.2.4 Growth of anodic electrocatalytic biofilms ......................................................................................66

4.2.5 Cyclic voltammetry ..........................................................................................................................66

4.2.6 Electrochemical data processing ......................................................................................................66

4.2.7 Spectroelectrochemical set-up for SERRS measurements ...............................................................66

4.2.8 SERRS measurements ......................................................................................................................66

4.3 Results and discussion ..........................................................................................................................67

4.3.1 Bioelectrochemical current production ............................................................................................67

4.4 Conclusions ..........................................................................................................................................70

5.1 Introduction ..........................................................................................................................................71

5.2 Materials and methods ..........................................................................................................................73

5.2.1 Carbon fiber preparation ..................................................................................................................73

5.2.2 Electrode preparation .......................................................................................................................75

5.2.3 Bioelectrochemical experiments ......................................................................................................75

5.3 Results and discussion ..........................................................................................................................76

5.3.1 Biocatalytic current generation at modified carbon electrodes ........................................................76

5.3.2 Analysis of electroactive biofilms grown at modified carbon electrodes with Scanning electron

microscopy ....................................................................................................................................................77

5.3.3 Cyclic voltammetry of electroactive biofilms grown at modified carbon electrodes .......................79

5.4 Conclusions ..........................................................................................................................................81

Page 12: PhD Thesis Alessandro Carmona2012

-v-

6 Electrospun carbon fiber mat with layered architecture for anode in microbial fuel

cells...........................................................................................................................................82

7 Electroactive mixed culture derived biofilms in microbial bioelectrochemical

systems: the role of pH on biofilm formation, performance and composition ................. 90

7.2.8.1 Flow-cytometry .......................................................................................................................94

7.2.8.1.1 Sample fixation and DNA staining .................................................................................... 94

7.2.8.1.2 Multiparametric flow-cytometry ........................................................................................ 94

7.2.8.2 T-RFLP and Sequencing .........................................................................................................95

6.1 Introduction ..........................................................................................................................................82

6.2 Materials and methods ..........................................................................................................................83

6.2.1 Carbon fiber preparation ..................................................................................................................83

6.2.2 Electrode preparation .......................................................................................................................84

6.2.3 Bioelectrochemical measurements ...................................................................................................84

6.2.4 SEM imaging ...................................................................................................................................84

6.3 Results and discussion ..........................................................................................................................85

6.3.1 Properties and performance of carbon fiber mat electrode materials ...............................................85

6.3.2 Biocatalytic current generation at carbon fiber mat electrode materials ..........................................87

6.3.3 Analysis of electroactive biofilms grown at carbon fiber mat electrode materials with Scanning

electron microscopy ......................................................................................................................................87

6.4 Conclusions ..........................................................................................................................................89

7.1 Introduction ..........................................................................................................................................90

7.2 Materials and methods ..........................................................................................................................91

7.2.1 General conditions ...........................................................................................................................91

7.2.2 Electrochemical set-up .....................................................................................................................92

7.2.3 Microbial inoculum and growth medium .........................................................................................92

7.2.4 Biofilm growth (fed-batch experiments) ..........................................................................................92

7.2.5 Biomass determination .....................................................................................................................93

7.2.6 Metabolic analysis ............................................................................................................................93

7.2.7 Continuous flow mode operation and pH-regime studies ................................................................93

7.2.8 Microbiological analysis ..................................................................................................................94

7.3 Results and discussion ..........................................................................................................................96

7.3.1 Biofilm formation and performance at different constant pH ..........................................................96

7.3.2 Biofilm performance at varying pH-environment during operation .................................................97

7.3.3 Influence of the pH and buffer capacity on the electron transfer .....................................................99

7.3.4 Microbial biofilm analysis .............................................................................................................101

7.4 Conclusions ........................................................................................................................................107

Page 13: PhD Thesis Alessandro Carmona2012

-vi-

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

Page 14: PhD Thesis Alessandro Carmona2012

-vii-

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

Page 15: PhD Thesis Alessandro Carmona2012

-viii-

Figure 2-2 A) and B) CVs for non-turnover conditions for S. oneidensis WT and mutants using a scan rate of 1

mV s−1

; C and D) provide the respective baseline corrected curves. ...................................................... 39

Figure 2-3 A) and B) CVs for turnover conditions for S. oneidensis WT and mutants using a scan rate of 1 mV

s−1

. ........................................................................................................................................................... 40

Figure 2-4 Plot of the base line corrected height of the oxidation peak of redox-system I (Δi−0.2) as function of

the maximum chronoamperometric current density of the respective microbial culture. ....................... 42

Figure 2-5 Plot of the corrected turnover CV signal and the performed analysis on the example of S. oneidensis

MR-1. (Similar plots of all strains can be found in Fig. S9-8 and Fig. S9-9 in the Supplementary

Information for Chapter 2). ..................................................................................................................... 43

Figure 3-1 Representative chronoamperometric fed-batch cycles of S. putrefaciens at graphite electrodes; applied

potentials: -0.1, 0, +0.1, +0.2, +0.3 and +0.4 V vs. Ag/AgCl; CV measurements during turn-over (A)

and non turn-over (B) conditions respectively. ....................................................................................... 53

Figure 3-2 Chronoamperometric current density of S. putrefaciens as function of the applied electrode potential.

................................................................................................................................................................ 53

Figure 3-3 A) Representative cyclic voltammograms of S. putrefaciens for turn-over conditions and B)

respective first derivatives of the voltammetric curves; scan rate: 1 mV s-1

. .......................................... 55

Figure 3-4 A) Cyclic voltammograms for non turn-over conditions for S. putrefaciens using a scan rate of 1 mV

s−1

; B provides the respective baseline corrected curves. ........................................................................ 56

Figure 3-5 Plot of the base line corrected height (○) and area (□) of the oxidation and reduction peaks of redox-

system shown in Fig. 3-4 as function of the applied potential. For visual convenience, reduction peak

areas are shown as negative values. ........................................................................................................ 57

Figure 3-6 Maximum intensity projection of confocal laser scanning microscopy data sets showing Shewanella

putrefaciens biofilms grown on electrode surfaces at different applied potentials. A) -0.1 V, B) 0 V, C)

+0.1 V, D) +0.2 V, E) +0.3 V and F) +0.4 V; (all vs. Ag/AgCl). Colour allocation: reflection of

electrode – grey, nucleic acid stained bacteria – green. .......................................................................... 58

Figure 3-7 Biofilm quantification of Shewanella putrefaciens biofilms grown on electrode surfaces at different

applied potentials. ................................................................................................................................... 59

Figure 4-1 Principle representation of a BES operating in the DET mode (see below). Electrons derived from the

oxidation of the organic substrate catalyzed by the bacterial cell are shuttled to the electrode via OMCs.

................................................................................................................................................................ 62

Figure 4-2 Electrochemical half cell set-up under potentiostatic control. Insert shows a photograph of the

nanostructured silver ring working electrode. ......................................................................................... 65

Figure 4-3 Chronoamperometric curve of a biofilm formation using a silver ring electrode poised at +0.05 V in a

batch experiment using 18 mM sodium lactate as the substrate and S. putrefaciens cells as biocatalyst.67

Figure 4-4 A) CV of the active biofilm formed on a silver ring electrode under non-turnover conditions (i.e. in

the absence of the substrate sodium lactate) at a scan rate of 1 mV s-1

. B) Respective SOAS baseline

corrected curves. ..................................................................................................................................... 68

Page 16: PhD Thesis Alessandro Carmona2012

-ix-

Figure 4-5 SERR spectra of the reduced (upper spectrum) and oxidized (lower spectrum) OMCs, obtained at -

425 and 0 mV, respectively. The spectra were obtained with excitation at λ = 413 nm, laser power of 1

mW, and an acquisition time of 90 s. Potentials refer to the Ag/AgCl (KCl 3 M) reference electrode

(210 mV vs. SHE). .................................................................................................................................. 69

Figure 5-1 (A) Schematic drawing of an electrospinning setup (derived from ref. (Greiner and Wendorff, 2007)).

Solution blowing differs from electrospinning by the use of a high-speed nitrogen jet flow (230–250 m

s-1

) instead of a high voltage electric field to accelerate and stretch the polymer solution into a fibrous

form (Sinha-Ray, et al., 2010). (B) Electrochemical cell for the simultaneous study of different

electrode materials. ................................................................................................................................. 73

Figure 5-2 Biocatalytic current generation at a GES-CFM modified carbon electrode in a model semi-batch

experiment. The GES-CFM electrode was modified by a wastewater-derived secondary biofilm grown

in a half-cell experiment under potentiostatic control. The electrode potential was 0.2 V. .................... 77

Figure 5-3 Scanning electron microscopic images of (A) carbon felt, (B) an electroactive biofilm grown at

carbon felt, (C) GES-CFM, (D) an electroactive biofilm grown at GES-CFM, (E) high resolution image

of GESCFM illustrating the occurrence of inter-fibre junctions, and (F) crosssectional view of GES-

CFM electrode. ....................................................................................................................................... 78

Figure 5-4 Cyclic voltammograms of an electroactive biofilm grown at GESCFM. The voltammograms were

recorded under turnover conditions [in the presence of substrate (10 mM acetate), curve A], as well as

nonturnover conditions (the absence of substrate, curve B). The biofilm was a wastewater-derived

secondary biofilm grown at a potential of 0.2 V under potentiostatic control. The scan rate was 1 mV s-

1. .............................................................................................................................................................. 80

Figure 6-1 A) Top view and B) cross-sectional view SEM images of carbon mat from TP; C) EDX spectra of

NCP-based carbon fiber; D) top view and E) cross-sectional view SEM images of layered-ECFM; F)

cross-sectional view SEM image of 2D-ECFM. ..................................................................................... 86

Figure 6-2 Biocatalytic current generation curves of carbon fiber mats in a half-cell experiment measured at

room temperature. Arrows represent replacement of medium. ............................................................... 87

Figure 6-3 SEM images of biofilms in: A-C belong to layered-CFM; D and E belong to commercial carbon felt;

and F belongs to 2D-ECFM. ................................................................................................................... 88

Figure 7-1 Performance of electroactive biofilms grown and operated at different pH-values: Maximum current

densities (filled circles; derived from chronoamperometric fed-batch experiments at 0.2 V vs. Ag/

AgCl) and coulombic efficiencies (open squares) of primary, wastewater derived biofilms are shown.

The substrate was 10 mM acetate. .......................................................................................................... 96

Figure 7-2 A) Chronoamperometric current density changes (at 0.2 V vs. Ag/ AgCl) for a biofilm initially grown

at pH 7.0 in relation to variations of the growth medium pH (numbers indicate the respective pH-value

of operation); B) Steady state current densities at 0.2 V vs. Ag/ AgCl of biofilms grown at pH 8 (circles)

and pH 7.0 (squares) at varying medium pH (derived from experiments similar as shown in A)). ........ 98

Figure 7-3 Influence of the operational pH: Cyclic voltammograms obtained at different operation pH (using a

constant ionic strength of 50 mM) at a scan rate of 1 mV s-1

during non-turnover conditions for

wastewater derived, acetate-fed biofilm formed at pH 7.0. (For pH 6 to pH 8 steady-state CVs are

shown, for pH 5 the 3rd CV-curve). ..................................................................................................... 100

Page 17: PhD Thesis Alessandro Carmona2012

-x-

Figure 7-4 Bacterial community profiles of the inoculum and the successive media of the anode chamber of a pH

7 grown biofilm (electrode-set 2). The profile of the community is cytometrically determined by the

cells’ DNA content labelled with the A-T specific fluorescent dye DAPI and the cells’ forward scatter

behaviour (FSC). As a result fingerprint-like cytometric patterns emerged as subsets of cells which

gather in numerous clusters of changing cell abundances therein. Up to 250000 cells were analysed and

the dominant sub-populations presented in yellow colour. The peak in the lower left corner of the

histograms represents the noise of the cytometer as well as unstained cell debris. ............................... 103

Figure 7-5 Dalmatian-n-MDS analysis with overlaid cytometric flow-plots derived from anode chamber

communities and anode biofilms when treated over several feeding cycles and different pH-values.

Black patches in flow-plots depict gate positions, cycle number is given with c 1–5 and pH-affiliation

with various grey/black labels (black: pH 7, grey: pH 9, light grey: pH 6, bold fringe around flow-plot:

electrodes; details see text and S11-2 to S11-10 for raw data). ............................................................. 106

Figure 8-1 A) Electrochemical half cell set-up under potentiostatic control and B) Exemplary established

bioelectrochemical active biofilm enriched from primary wastewater fed with acetate. The red color is

mainly caused by the hemes (Jensen, et al., 2010). ............................................................................... 112

Figure 8-2 Bioelectrocatalytic performance of electroactive microbial biofilms derived from different inocula

with fed batch operation in potentiostatically controlled half-cell experiments (+0.2 V vs. Ag/ AgCl) at

graphite rod electrodes. The substrate was 10 mM sodium acetate or sodium lactate respectively. ..... 114

Figure 8-3 Exemplary cyclic voltammograms. Electroactive microbial biofilms derived from different inocula

grown with Sodium acetate (10 mM) recorded during non-turnover (A, C, E and G) and turnover

conditions (B, D, F and H) conditions. The scan rate used was 1 mV s-1

. ............................................ 116

Figure 8-4 Exemplary cyclic voltammograms. Electroactive microbial biofilms derived from different inocula

grown with Sodium lactate (10 mM) recorded during non-turnover (A, C, E and G) and turnover

conditions (B, D, F and H) conditions. The scan rate used was 1 mV s-1

. ............................................ 117

Figure 8-5 Exemplary cyclic voltammograms from electroactive microbial biofilms derived from primary

wastewater grown with 10 mM sodium lactate (A) or 10 mM sodium acetate (B) recorded during

turnover conditions. First derivatives of biofilms grown with sodium lactate (C) or sodium acetate (D).

.............................................................................................................................................................. 118

Figure S9-1 Schematic drawing of an electrochemical cell for the study of the electron transfer mechanisms and

current production. The electrochemical cell consists of an anode, a cathode and, a membrane

separating both. An oxidation process occurs at the anode, in this case lactate oxidation, in which

electrons and protons are produced. The electrons flow to the cathode through an external circuit or

potentiostat in which the electrons can be can be quantified. Meanwhile the protons are released to the

media and lately they migrate to the cathode chamber to react with molecules of water and electrons

finally producing hydrogen for example. Figure drawn with modifications after (Rabaey and Verstraete,

2005, Schröder, 2008). .......................................................................................................................... 121

Figure S9-2 Electrochemical half cell set-up under potentiostatic control. Description: “Top view” shows the 5

necks of the 250 mL flask. In section A-A’ details of the working electrode, counter shielded electrode

and reference electrode are given. In section B-B’ the port for filtrated air, filtrated nitrogen and media

supply are detailed. ............................................................................................................................... 122

Page 18: PhD Thesis Alessandro Carmona2012

-xi-

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

Page 19: PhD Thesis Alessandro Carmona2012

-xii-

Figure S10-3 Effect of the Riboflavin concentration in the extracellular electron transfer. Representative cyclic

voltammogram of a Shewanella putrefaciens biofilm grown at a poised (+0.4 vs Ag/AgCl) graphite

electrode. The basal concentration of Riboflavin in the growth media was 0.1 μM as reported in the

Materials and Methods section (left panel). The voltammogram was recorded at maximum biofilm

activity after the start of the chronoamperometry with a scan rate of 1 mV s-1

. Voltammetry of all

Shewanella biofilms grown at different applied potentials with no additional supplementation of

Riboflavin (0.1 μM) showed only one inflection point centered at 0 V (vs Ag/AgCl). After six semi

batch chronoamperometric cycles a pulse of fresh substrate containing 1 μM of Riboflavin was injected

into the electrochemical cell (right panel). For the experiment with additional Riboflavin (1 μM) not

only the inflection point at 0 V was observed but also an inflection point centered at -0.4 V

characteristic of the mediator molecule Riboflavin (Peng, et al., 2010b), indicating that this molecule

participated in the extracellular electron transfer process. Furthermore, from the pronounced sharp rise

of the inflection point centered at the midpoint potential of Riboflavin, provided an example of how this

mediator molecule increases the electron transfer (Marsili, et al., 2008a). ........................................... 135

Figure S11-1 Influence of the buffer capacity: Cyclic voltammogramms (1mV s-1

) at pH 7, wastewater derived

and acetate–fed biofilms at varying buffer concentration, A) non-turn over B) turn over conditions. . 136

Figure S11-2 T-RFLP chromatograms (restriction digestion with RsaI and MspI) of the anode biofilms formed at

pH 7. The x axis represents the length of terminal restriction fragments and the y axis the relative

fluorescence units. On the right the area of every peak is shown as percentage of the total peak area. The

RsaI peak at 238 bp (503 bp with MspI) is shown in bright yellow and represents Geobacter

sulfurreducens (identified after sequencing). ........................................................................................ 137

Figure S11-3 T-RFLP chromatograms (restriction digestion with RsaI and MspI) of the anode biofilms formed at

pH 9. The x axis represents the length of terminal restriction fragments and the y axis the relative

fluorescence units. On the right the area of every peak is shown as percentage of the total peak area. The

peak at 238 bp (503 bp with MspI) is shown in bright yellow and represents Geobacter sulfurreducens

(identified after sequencing). In the sample of electrode-set 2 this organism could not be detected. This

biofilm comprised several phylotypes. ................................................................................................. 138

Figure S11-4 T-RFLP chromatograms (restriction digestion with RsaI and MspI) of the anode biofilms formed at

pH 6. The x axis represents the length of terminal restriction fragments and the y axis the relative

fluorescence units. On the right the area of every peak is shown as percentage of the total peak area. The

RsaI peak at 238 bp in the electrode-set 2 is shown in bright yellow and represents Geobacter

sulfurreducens (identified after sequencing the sample of electrode-set 2). In the small dashed window

the peak position is drawn to a larger scale to see that the peak position of the RsaI peak is different in

the sample of set 1 and set 2. The main MspI peak is found at 161 bp that is also different from what

was found for Geobacter sulfurreducens in the other samples (Figures S11-2 and S11-3 above). This

clearly shows that Geobacter sulfurreducens could not be detected in the sample of electrode-set 1. This

biofilm comprised several phylotypes. ................................................................................................. 139

Page 20: PhD Thesis Alessandro Carmona2012

-xiii-

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

Page 21: PhD Thesis Alessandro Carmona2012

-xiv-

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

Page 22: PhD Thesis Alessandro Carmona2012

- 1 -

CHAPTER I

1 Extracellular electron transfer in Bioelectrochemical

systems: bridge between natural environments and

applied technologies

1.1 Prelude

In this introductory chapter a comprehensive description of microbial electron transfer

mechanisms in anoxic natural environments and the application of this natural process into a

promising, multi interdisciplinary -and still in continuing development technology- is given.

Section 1.2 illustrates the ecological significance of insoluble metal electron acceptors in nature.

Iron is taken as a model example to explain its bio-mobility in the environment. Here the

participation of some exemplary microorganisms capable of reducing iron is described. Section

1.3 provides a general definition of microbial extracellular electron transfer (ET) and describes

how microbiologists discovered this process in two model microorganisms now commonly used

as exemplary dissimilatory metal reducing bacteria. Later, one of the first applications for ET in

the field of bio-remediation and more recently in the field of Bioelectrochemical systems (BESs)

is provided. BESs not only have allowed the study of microbial ET but also permitted the

development of promising applications. Section 1.4 presents two known ET mechanisms

performed by bacteria, i.e., direct and mediated extracellular electron transfer (DET and MET

respectively). For DET, detailed descriptions on representative dissimilatory metal reducing

bacteria are given. In the case of MET, mediating redox species that transfer electrons between

the bacteria and the final electron acceptor are presented. Section 1.5 gives an overall

introduction to BESs. First, BESs represent an additional approach for the study of microbial ET

and second, they have emerged as an applied technology based on microbial ET. Finally Section

1.6 provides a comprehensive view on one of the main motivations in the development of BESs:

the improvement of current density production focused for near future applications. Different

aspects are exemplified with the case of 3D new electrode materials that improve the overall

performance of BESs. Finally, several environmental factors affecting the formation and

performance of electroactive biofilms are discussed.

Page 23: PhD Thesis Alessandro Carmona2012

-2-

1.2 Ecological significance of insoluble metal electron acceptors: the example of iron

Until the late 70s, reduction of Fe(III) to Fe(II) in sedimentary and subsurface environments was

believed to be the result of purely abiotic processes (Cornell and Schwertmann, 2007, Fenchel

and Blackburn, 1979). Now it is known that bacterial utilization of Fe(III) oxides as the terminal

electron acceptor is an important practice in anaerobic environments in which the reduction of

Fe(III) to Fe(II) is a enzymatically catalyzed bacterial process (Gralnick and Newman, 2007,

Lovley, 1993). Bacterial reduction of Fe(III) oxides has diverse significant ecological

repercussions, for example the quality of water can be modified by the increment of dissolved

Fe(II) that changes the taste of drinking water (Lovley, 2000) and furthermore Fe(III) is thought

to be the most abundant of all the available terminal electron acceptors in several subsurface

environments (Lovley, 1991). Some known representative microorganisms capable of utilizing

iron as final electron acceptor include: Geobacter metallireducens (Lovley, 1993),

Desulfuromonas acetoxidans (Roden and Lovley, 1993), Pelobacter carbinolicus (Lovley, et al.,

1995), members of the genus Desulfuromusa (Fredrickson and Gorby, 1996), Shewanella

oneidensis (Myers and Nealson, 1988), Ferrimonas balearica (Lovley, 2000), Geovibrio

ferrireducens (Caccavo Jr, et al., 1996) and Geothrix fermentans (Coates, et al., 1999).

The reduction of Fe(III) is considered as a predominant process due to the iron cycle reactions

(Lovley, et al., 1993), some of them with an important participation of bacteria (see below).

According to Luu and Ramsay (Luu and Ramsay, 2003), first solid oxides settle into the oxygen

transition zone called suboxic zone (Fig. 1-1). Simultaneously phosphate and metals are

removed via precipitation and complexation. In the suboxic zone carbon oxidation takes place

by bacteria via the utilization of iron as terminal electron acceptor. During iron reduction,

organic phosphate and metals are released into the oxic zone. From the oxidation of carbon,

Fe(II) forms insoluble precipitates in the suboxic zone such as siderite (FeCO3), pyrite (FeS2),

vivianite [Fe3(PO4)2] and magnetite (Fe3O4). Additionally some species of Fe(II) diffuse into the

oxic zone where finally reoxidation of Fe(II) occurs to form insoluble oxides and if no input of

organic carbon takes place, oxides accumulate in sediments of the suboxic zone, otherwise the

cycle continues again. Since the distribution of Fe(III) in the environment depends on the

amount of organic matter present (Pan, et al., 2011), Fe(III) oxides get retained in the sediment

when no organic matter is available diminishing the cycling of iron. Therefore the mobility of

certain compounds in the environment mainly depends on the biotransformation of organic

matter by microorganisms, making the study of these processes of great importance.

Page 24: PhD Thesis Alessandro Carmona2012

-3-

Figure 1-1 Simplified iron cycle in aquatic environments. Figure drawn with modifications after

(Luu and Ramsay, 2003, Nealson and Saffarini, 1994).

1.3 Electron transfer processes in the environment

Extracellular electron transfer (ET) is a general mechanism by which microorganisms generate

energy for cell growth and maintenance (Hernandez and Newman, 2001), i.e., bacteria transfer

electrons from their internal metabolism through a chain of trans-membrane proteins to finally

reduce insoluble metal electron acceptors. In the early 90s, environmental microbiologists

realized the importance of microbial ET to insoluble metal electron acceptors in several

biogeochemical cycles and progressively applied this extraordinary finding, e.g., on the

bioremediation of contaminated sites (Lovley, 1991, Nealson, et al., 1991). More recently this

finding has been used in an interdisciplinary way not only to study the fundamentals of

microbial ET but also to apply this concept in the so-called Bioelectrochemical systems (BESs)

(Rabaey, et al., 2010) (section 1.5). The basic and applied interest on microbial ET has rapidly

increased since the publication of two breakthrough papers introducing two of the first known

bacteria capable of reducing insoluble metal electron acceptors: Shewanella oneidensis MR-1

(Myers and Nealson, 1988) and Geobacter sulfurreducens PCA (Caccavo, et al., 1994).

Page 25: PhD Thesis Alessandro Carmona2012

-4-

Furthermore, the exploration of how microbes breathe minerals has been later stimulated by the

publication of both genomes (Heidelberg, et al., 2002, Methé, et al., 2003), making possible

genetic manipulations to study their respective ET pathways (see Chapter 2 for an example on

Shewanella oneidensis MR-1 knock-out mutants).

1.4 Microbial extracellular electron transfer mechanisms

To date mainly two microbial ET mechanisms have been recognized in the literature (Gralnick

and Newman, 2007, Hernandez and Newman, 2001, Lovley, 2011, Schröder, 2007, Watanabe,

et al., 2009). In one of those mechanisms named as direct extracellular electron transfer (DET),

electrons are transferred from the respiratory chain in the cell to an extracellular insoluble

compound or final electron acceptor (e.g., iron oxides or conductive electrode materials in

BESs) via a complex architecture involving several outer membrane cytochromes (Millo, et al.,

2011) (Fig 1-2A), an ability often conventionally awarded only to gram-negative bacteria

(Hernandez and Newman, 2001, Lovley, 2008a, Rosenbaum, et al., 2011, Shi, et al., 2009) with

some recent exceptions of gram-positive bacteria (Cournet, et al., 2010, Marshall and May,

2009, Wrighton, et al., 2011).

Figure 1-2 Overall illustration of microbial ET mechanisms found in the literature. A) Direct

extracellular electron transfer via membrane bound cytochromes and conductive nanowires and

B) Mediated extracellular electron transfer via a mediator molecule (Medred

or Medox

) (see text).

Here ET mechanisms are represented in the field of BESs with electrode materials as final

electron acceptors but the same illustration could be applied for bacteria in natural environments

using for instance iron oxides as terminal electron acceptors. Figure drawn with modifications

after (Schröder, 2007).

Page 26: PhD Thesis Alessandro Carmona2012

-5-

Another well-considered DET mechanism which is still under investigation is the ET via

cellular appendages facing the extracellular environment (i.e., microbial nanowires) found in

several bacteria (Bretschger, et al., 2010b) (Fig 1-2A) (see section 1.4.1). On the other side,

microorganisms are capable of ET via mediator molecules that, i) get reduced by outer

membrane cytochromes and later oxidized onto extracellular insoluble compounds or onto

conductive electrode materials as in the case of BESs; or ii) via periplasmatic or cytoplasmatic

redox couples that serve as reversible terminal electron acceptors, transferring electrons from the

bacterial cell to a final electron acceptor (Schröder, 2007). This mechanism is usually named as

mediated extracellular electron transfer (MET) (Marsili and Zhang, 2010) (Fig 1-2B) (see

section 1.4.2).

1.4.1 Microbial direct extracellular electron transfer (DET)

1.4.1.1 DET via membrane-bound redox-enzymes

As pointed out in section 1.2, diverse groups of microorganisms are now known to engage in

electron transfer to extracellular insoluble compounds. More recently with the use of conductive

electrode materials (anodes) in BESs, an additional number of microorganisms have joined to

the list of -recently named- exoelectrogenic bacteria capable of performing DET (Logan, 2009);

e.g., Desulfuromonas acetoxidans (Bond, et al., 2002), Escherichia coli K12 (Schröder, et al.,

2003), Rhodoferax ferrireducens (Chaudhuri and Lovley, 2003), Aeromonas hydrophila (Pham,

et al., 2003), Desulfobulbus propionicus (Holmes, et al., 2004a), Hansenula anomala (Prasad, et

al., 2007), Rhodopseudomonas palustris DX-1 (Xing, et al., 2008), Klebsiella pneumoniae L17

(Zhang, et al., 2008) and Proteus vulgaris (Rawson, et al., 2011) among others. While it is

commonly accepted that microbial ET occurs within complex communities found in BES

anodes (Logan and Regan, 2006a), the in-depth study of microbial ET mechanisms has revolved

around two model exoelectrogenic bacteria families: Shewanellaceae and Geobacteraceae

(Bretschger, et al., 2010b).

1.4.1.1.1 Shewanella oneidensis DET via membrane-bound redox-enzymes

As recently reported by Shi and co-workers (Shi, et al., 2009), DET performed by Shewanella

oneidensis depends on inner (IM) and outer membrane (OM) proteins that are known to be

directly involved in the reduction of insoluble metals that act as extracellular electron acceptors

(or in the case of BESs: electrode materials). These proteins include the inner membrane

tetrahaem c-Cyt CymA that is a homologue of NapC/NirT family of quinol dehydrogenases, the

Page 27: PhD Thesis Alessandro Carmona2012

-6-

periplasmic decahaem c-Cyt MtrA, the outer membrane protein MtrB and the OM decahaem c-

Cyts MtrC and OmcA (Fig. 1-3A).

All these proteins together form a pathway to transfer electrons from the quinone/quinol pool in

the inner membrane to the periplasm (PS) and then to the outer membrane where MtrC and

OmcA can transfer electrons directly to the surface of electrode materials.

1.4.1.1.2 Geobacter sulfurreducens DET via membrane-bound redox-enzymes

On the other side, DET performed by Geobacter sulfurreducens (as reported by Shi and co-

workers (Shi, et al., 2009)) relies on the outer membrane proteins tetrahaem c-Cyt OmcE and

hexahaem c-Cyt OmcS that are believed to be located on the cell surface where they are

suggested to transfer electrons to type IV pili. Type IV pili are hypothesized to transfer electrons

directly to Fe(III) oxides (or in the case of BESs: electrode materials). OmcE and OmcS also

receive the electrons from the quinone/quinol pool in the inner membrane (Fig. 1-3B).

Figure 1-3 Roles of outer membrane cytochromes of A) Shewanella oneidensis and B)

Geobacter sulfurreducens in extracellular electron transfer. IM: inner membrane, OM: outer

membrane and PS: periplasm. Figure drawn with modifications after (Shi, et al., 2009).

1.4.1.2 DET via self-produced microbial nanowires

The fundamental comprehension of microbial ET mechanisms is still in progress (Bretschger, et

al., 2010b) since non-conclusive and debatable experimental evidence of an additional DET

process via self-produced microbial nanowires has come to light (Lovley, 2011). This recently

found DET mechanism is not only expected to change the way scientists will look at microbial-

Page 28: PhD Thesis Alessandro Carmona2012

-7-

electrode interactions but also it could commence a new whole applied research field due to the

promising application of microbial nanowires as nano bio-conductive materials (Malvankar, et

al., 2011). In general, the information devoted to the analysis of conductive bacterial nanowires

is scarce. However experimental evidence of microbial-like nanowires has been reported for

some microorganisms as described below. There exists evidence showing the presence of

microbial-like nanowires in nutrient limited cultures of the cyanobacterium Synechocystis sp.

PCC 6803 (Fig. 1-4C) and in co-cultures of Pelotomaculum thermopropionicum and

Methanothermobacter thermautotrophicus (Fig. 1-4D) (Gorby, et al., 2006). Additionally,

putative nanowires have been observed in sulfate limiting cultures of Desulfovibrio vulgaris and

in environmental samples from hydrothermal vents. Nevertheless, only visual information in

this regard has been presented so far (Bretschger, et al., 2010b). Whereas microbial-like

nanowires structures have been observed in several bacterial cultures (Bretschger, et al., 2010b),

hitherto; to the best of my knowledge and beyond the optical description, only four works

devoted to the electrochemical and spectroscopical characterization of these structures have

been published (according to “Scopus”, February 2012) and all of them using either the model

exoelectrogenic bacterium G. sulfurreducens or S. oneidensis.

1.4.1.2.1 Geobacter sulfurreducens DET via self-produced microbial nanowires

One of the first observations on microbial nanowires was made by Reguera and co-workers

(Reguera, et al., 2005) on G. sulfurreducens. They have found that a nanowire-deficient mutant

of G. sulfurreducens could not reduce Fe(III). Additionally by using atomic force microscopy

they suggested that these G. sulfurreducens nanowires could be conductive. A few years later,

additional information on the possible conductivity of G. sulfurreducens nanowires was

provided by Malvankar and co-workers (Malvankar, et al., 2011). They have showed the

metallic-like conductivity (along centimeter-length scale) in microbial nanowires produced by

G. sulfurreducens. Moreover, they have even suggested that these structures could possess

similar properties to those of synthetic metallic nanostructures (Fig. 1-4A).

1.4.1.2.2 Shewanella oneidensis DET via self-produced microbial nanowires

On the other hand, only one year later to the first finding of nanowires in G. sulfurreducens,

Gorby and co-workers provided evidence on the conductivity of electrical microbial nanowires

produced by S. oneidensis in direct response to electron-acceptor limitations (Gorby, et al.,

2006). Four years later El-Naggar and co-workers (El-Naggar, et al., 2010) presented an

additional contribution in this regard confirming the conductivity of such microbial nanowires

produced by S. oneidensis MR-1 (Fig. 1-4B). Independent of the source of microbial nanowires,

Page 29: PhD Thesis Alessandro Carmona2012

-8-

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.

Page 30: PhD Thesis Alessandro Carmona2012

-9-

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

Page 31: PhD Thesis Alessandro Carmona2012

-10-

1.5) has stimulated the general interest on externally microbial ET (Bond, et al., 2002, Kim, et

al., 1999a).

To date, mainly experiments with gram-negative bacteria have contributed with evidence that

microorganisms are able to perform MET mechanisms (Marsili and Zhang, 2010). Microbial

known mediators are listed in Table 1-1. In general, these molecules have provided experimental

evidence on the possibility to transfer electrons to electrode materials and according to

assumptions made by Marsili and Zhang (Marsili and Zhang, 2010), redox mediator molecules

would be able to transfer electrons between bacteria and final electron acceptors regardless of a

solid metal oxide or an electrode material. Such an ability in conjunction with the fact that self-

produced mediator molecules from one bacteria can be used further by a different bacteria (as in

the case of Pseudomonas sp. and Brevibacillus sp. PTH1 (Pham, et al., 2008)) increases the

applications of this specific MET mechanism.

Table 1-1 Representative microbially produced redox mediators.

Microoganism Mediator molecule Reference

Sphingomonas xenophaga 4-amino-1,2-naphthoquinone

(Keck, et al., 2002)

Pseudomonas aeruginosa Phenazine-1-carboxylic acid

(Price-Whelan, et al., 2006)

Pseudomonas chlororaphis Phenazine-1-carboxamide

(van Rij, et al., 2004)

Shewanella oneidensis Flavin mononucleotide

(von Canstein, et al., 2008)

Shewanella algae Melanin

(Turick, et al., 2002)

Bacillus pyocyaneus Pyocyanine

(Friedheim and Michaelis,

1931)

Propionibacterium

freundenreichii

2-Amino-3-carboxy-1,4-

naphthoquinone

(Hernandez and Newman,

2001)

Shewanella alga Cyanocobalamin

(Workman, et al., 1997)

Acetobacterium woodii Hydroxycobalamin (Hashsham and Freedman,

1999)

Pseudomonas stutzeri Pyridine-2,6-bis

(Lewis, et al., 2001)

Methanosarcina thermophila Porphorinogen-type molecules

(Koons, et al., 2001)

Geobacter metallireducens Anthraquinone-2,6-disulfonate

(Cervantes, et al., 2004)

Shewanella oneidensis 1,4-Dihydroxy-2-naphthoate

derivative (Ward, et al., 2004)

Klebsiella pneumoniae Anthraquinone-2,6-disulfonate

(Li, et al., 2009b)

aMore detailed information can be found in the following references: (Hernandez and Newman,

2001, Marsili and Zhang, 2010, Schröder, 2007, Watanabe, et al., 2009).

Page 32: PhD Thesis Alessandro Carmona2012

-11-

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

Page 33: PhD Thesis Alessandro Carmona2012

-12-

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.

Page 34: PhD Thesis Alessandro Carmona2012

-13-

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).

Page 35: PhD Thesis Alessandro Carmona2012

-14-

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).

Page 36: PhD Thesis Alessandro Carmona2012

-15-

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.

Page 37: PhD Thesis Alessandro Carmona2012

-16-

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

Page 38: PhD Thesis Alessandro Carmona2012

-17-

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

Page 39: PhD Thesis Alessandro Carmona2012

-18-

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

Page 40: PhD Thesis Alessandro Carmona2012

-19-

substantial improvements (10 to 100 fold) in current and power densities in comparison to

conventional two-dimensional materials (Schröder, 2011). More recently Katuri and co-workers

(Katuri, et al., 2011) fabricated three-dimensional microchannelled nanocomposite electrodes.

These materials allowed the growth of Geobacter sulfurreducens biofilms over the three-

dimensional surface, providing acetate oxidation current densities of up to 25 A m−2

. Xie and

co-workers (Xie, et al., 2011) reported a carbon nanotube sponge composite that provided a

three-dimensional scaffold that was favorable for microbial colonization. This nanotube sponge

allowed the increment of 2.5 times the previously reported maximum areal power density and 12

times the previously reported maximum volumetric power density.

Independently from the previous examples on carbon based three dimensional electrode

materials, Chapter 5 and 6 present two studies in this regard showing conditions that allowed the

production of the highest current density values reported so far by bio-electrochemically active

biofilms.

1.6.2 Performance based on the study of environmental factors affecting biofilm

formation

In the field of BESs, it has been assumed that the treatment of wastewater could be one of the

most appealing applications (Logan, et al., 2006). In fact, in order to make BESs a successful

technology in wastewater treatment, researchers have to pay special attention to the

environmental and external factors that influence the biofilm, considered to be “powerhouse” of

BESs (Franks, et al., 2010). In the literature one can find different approaches that have been

utilized in order to decipher the factors influencing the formation of anodic biofilms in BESs.

For instance, Patil and co-workers (Patil, et al., 2010) investigated the temperature dependence

and temperature limits of wastewater derived anodic microbial biofilms. They demonstrated that

these biofilms are active in a temperature range between 5 and 45°C. Additionally, they also

demonstrated that elevated temperatures during initial biofilm growth not only accelerated the

biofilm formation process but they also influenced the bioelectrocatalytic performance of these

biofilms when measured at identical operation temperatures. For example, the time required for

biofilm formation decreased from above 40 days at 15°C to 3.5 days at 35°C. On the other side,

Zhang and co-workers (Zhang, et al., 2011) investigated the effects of external resistance on

biofilm formation and electricity generation of microbial fuel cells. The morphology and

structure of the biofilms developed at 10, 50, 250 and 1000 Ω was characterized. They

demonstrated that the biofilm structure played a crucial role in the maximum power density and

Page 41: PhD Thesis Alessandro Carmona2012

-20-

sustainable current generation of BESs. Their results showed that, maximum power density of

their BESs increased when the external resistance decreased. They have attributed their results

to the existence of void spaces beneficial for proton and buffer transport within the anode

biofilm, which maintains a suitable microenvironment for electrochemically active

microorganisms. Furthermore, Biffinger and co-workers (Biffinger, et al., 2009) used a high-

throughput voltage based screening assay to correlate current output from a BESs containing

Shewanella oneidensis MR-1 to biofilm coverage over 250 h (among other experimental

conditions). BESs operated by Biffinger and co-workers permitted data collection from nine

simultaneous S. oneidensis MR-1 BESs experiments in which each experiment was able to

demonstrate organic carbon source utilization and oxygen dependent biofilm formation on a

carbon electrode. Finally, Ieropoulos and co-workers (Ieropoulos, et al., 2010) have

hypothesized that the processing of large volumes of wastewater in BESs would require

anodophilic bacteria operating at high flow-rates. Therefore, they examined the effect of flow-

rate on different microbial consortia during anodic biofilm development using inocula designed

to enrich either aerobes/ facultative species anaerobes. By using scanning electron microscopy

they showed some variation in biofilm formation where clumpy growth was associated with

lower power. In a different category, experiments using genetic manipulations should be

mentioned. For example, the use of knocked mutants of bacteria in order to delete from their

genome the production of outer membrane surface structures needed to adhere to solid surfaces

and generate ticker and robust electroactive biofilms (Bouhenni, et al., 2010, Rollefson, et al.,

2009). Regardless of the previous examples on factors influencing the formation of anodic

biofilms in BESs, Chapter 7 and 8 present two more detailed examples on this aspect.

Page 42: PhD Thesis Alessandro Carmona2012

-21-

1.7 Aim of this Dissertation

Because of the issues raised in the previous sections in this chapter, the aim of my dissertation

was to investigate different aspects of microbial-electrode interactions in BESs. The different

objectives of this Ph.D. Thesis are divided into the following chapters:

Part I Electron transfer mechanisms of pure culture biofilms of Shewanella spp.

Chapter 2 Cyclic voltammetric analysis of the electron transfer of Shewanella

oneidensis MR-1 and nanofilament and cytochrome knock-out mutants.

Chapter 3 Study of Shewanella putrefaciens biofilms grown at different applied

potentials using cyclic voltammetry and confocal laser scanning microscopy.

Chapter 4 Spectroelectrochemical analysis of intact microbial biofilms of Shewanella

putrefaciens for sustainable energy production.

Part II Porous 3D carbon as anode materials for performance of electrochemically active mixed

culture biofilms.

Chapter 5 Electrospun and solution blown three-dimensional carbon fiber nonwovens

for application as electrodes in microbial fuel cells.

Chapter 6 Electrospun carbon fiber mat with layered architecture for anode in microbial

fuel cells.

Part III The influence of external factors on electrochemically active mixed culture biofilms.

Chapter 7 Electroactive mixed culture biofilms in microbial bioelectrochemical

systems: the role of pH on biofilm formation, performance and composition.

Chapter 8 Electroactive mixed culture biofilms in microbial bioelectrochemical

systems: the role of the inoculum and substrate on biofilm formation, performance and

composition.

Page 43: PhD Thesis Alessandro Carmona2012

-22-

1.8 Structure of the Thesis and personal contribution

This Ph.D. Thesis tackled various aspects within bioelectrochemical systems. For this reason,

several available experimental techniques were utilized. From electrochemical voltammetric

techniques, via confocal laser scanning microscopy, to surface-enhanced resonance Raman

scattering. The results of this Thesis are divided into three main parts regarding the respective

area of study:

Part I Electron transfer mechanisms of pure culture biofilms of Shewanella spp.

Part II Porous 3D carbon as anode materials for performance of electrochemically active mixed

culture biofilms.

Part III The influence of external factors on electrochemically active mixed culture biofilms.

From Fig. 1-8 one can see that the different parts of this Thesis were focused mainly on the

investigation of several processes occurring at the interface between the electrode material and

bioelectroactive biofilms. In the following lines, the chapters contained in this thesis are listed.

Furthermore an appreciation of my personal contribution to each is provided.

Figure 1-8 Schematic illustration of the research areas within the three chapter I, II and III.

Page 44: PhD Thesis Alessandro Carmona2012

-23-

Part I. Electron transfer mechanisms of pure culture

biofilms of Shewanella spp.

CHAPTER 2

The idea of this project came as a continuation of previous experiments performed on

Shewanella at the University of Greifswald. The reason to use Shewanella oneidensis MR-1 as a

biological model came from a collaboration impelled by Dr. B.R. Ringeisen’s visit to our

Institute in November 2008. At that time Prof. Dr. U. Schröder and Dr. F. Harnisch motivated

me to work on electrochemical active biofilms of wild-type and mutant strains of S. oneidensis

kindly provided by Dr. B.R. Ringeisen’s team at the US Naval Research Laboratory. The

growth of Shewanella strains was performed in close collaboration with L.A. Fitzgerald and J.C.

Biffinger. I designed, planned and performed all experimental work in Braunschweig in close

collaboration with Dr. F. Harnisch. I analyzed/ interpreted data and wrote the manuscript

together with Dr. F. Harnisch. During the whole process of this project we maintained useful

discussions with Dr. B.R. Ringeisen’s team. Prof. Dr. U. Schröder gave useful advice.

CHAPTER 3

The lack of information on the electron transfer mechanisms of electrochemical active biofilms

of Shewanella initiated this study. Most of the previous studies were generally carried out with a

single applied potential or each study used different operational parameters, which makes it

difficult to compare among studies. I took care of the whole maintenance process and growth of

S. putrefaciens. I designed, planned and performed all experimental work in Braunschweig in

close collaboration with Dr. F. Harnisch and Prof. Dr. U. Schröder. CLSM measurements were

performed at the Helmholtz Centre, Magdeburg by U. Kuhlicke and me in close collaboration

with Dr. T.R. Neu. I analyzed/ interpreted data and wrote the manuscript together with Dr. F.

Harnisch. For the CLSM data we maintained invaluable discussions with Dr. T.R. Neu. Prof.

Dr. U. Schröder gave useful advice and guidance.

Page 45: PhD Thesis Alessandro Carmona2012

-24-

CHAPTER 4

The idea of this project came as a continuation of previous spectroelectrochemical experiments

performed in our group on Geobacter biofilms. The idea to use Shewanella putrefaciens as a

biological model came from the total lack of information on the electron transfer mechanisms

using spectroelectrochemical tools such as surface-enhanced resonance Raman scattering. This

project was conducted in several phases at the TU Braunschweig and at the TU Berlin. At the

TU Braunschweig, I took care of the whole maintenance process and growth of S. putrefaciens

biofilms in electrochemical half-cells. In close collaboration with Dr. D. Millo, I designed,

planned and performed experimental work at the TU Berlin in the group of Prof. Dr. P.

Hildebrandt. Under the supervision of Dr. Millo (who performed the SERRS experiments and

analyzed the spectra with the assistance of Khoa H. Ly), and Dr. Harnisch, I analyzed and

interpreted data. During the whole process of this project, Prof. Dr. U. Schröder has given useful

advice and guidance.

Part II. Porous 3D carbon as anode materials for

performance of electrochemically active mixed culture

biofilms

CHAPTER 5

The motivation of the study can be assigned to the continuous efforts in the field of BESs to

improve the overall performance of the systems; especially in terms of electrode materials, as

the bioelectrocatalytic activity plays a key role. The motivation for this project came from S.

Chen who was finishing at the time his Ph.D. at the Philipps-Universität in Marburg in the group

of Prof. Greiner. This project was conducted in several phases at the Universty of Marburg and

the TU Braunschweig. At the TU Braunschweig we tested a series of 3D porous carbon fiber

based materialss, produced by gas-assisted electrospinning at the Philipps-Universität and a

series of electrospun and solution-blown carbon fibers fabricated by the group of Prof. Yarin at

the University Illinois at Chicago on the suitability to serve as electrode materials for BESs. At

all times I was deeply involved in the growth and maintenance of waste-water derived

electroactive biofilms and the test of the mentioned electrode materials as well as data analysis

and interpretation.

Page 46: PhD Thesis Alessandro Carmona2012

-25-

CHAPTER 6

Encouraged by our previous work presented in detail in Chapter 5, this study was driven by the

continuous efforts in the field of BESs to develop high performance three-dimensional electrode

materials for electroactive biofilms. This project was conducted at the Philipps-Universität

Marburg in the group of Prof. Greiner (material development) and at the TU Braunschweig in

the group of Prof. Schröder (electrode characterization). We tested a series of electrospun

carbon fiber mats with layered architecture and investigated these materials on their suitability

for growth and performance of electroactive waste water derived anodic biofilms. At all times I

was deeply involved in the growth and maintenance of the waste-water derived electroactive

biofilms and the test of the mentioned electrode materials as well as data analysis and

interpretation.

Part III. The influence of external factors on

electrochemically active mixed culture biofilms

CHAPTER 7

The investigation of environmental parameters that affect the formation and performance of

electroactive biofilms stimulated this study since the majority of studies are restricted to a

neutral pH. Specifically how the pH value influences the biofilm growth (lag-time), steady state

anodic bioelectrocatalytic activity and microbial composition. I was involved in the replication

of fed-batch experiments at pH 6, 7 and 9 and also in the operation of continuous flow

experiments for pH-regime and buffer capacity studies.

CHAPTER 8

Most of the experiments designed to study electroactive biofilms in BESs are generally carried

out with one substrate or one microbial inoculum varying different operational parameters.

Therefore in order to exclude the influence of operational variables and to investigate only the

effect of an individual microbial inoculum source and an individual substrate, the experiments

here presented were conducted with half-cell set-ups under potentiostatic control with multiple

inocula and substrates. I was deeply involved in the recollection of inocula samples, preparation

of materials needed for half-cell experiments, later in the growth and maintenance of

electroactive biofilms and as well as in data collection, analysis and interpretation.

Page 47: PhD Thesis Alessandro Carmona2012

-26-

1.9 Comprehensive summary

Shewanella is frequently used as a model microorganism for microbial bioelectrochemical

systems (BESs) such as microbial fuel cells (MFCs) or microbial electrolysis cells (MECs). In

chapter 2, we used cyclic voltammetry (CV) to investigate extracellular electron transfer

mechanisms from Shewanella oneidensis MR-1 (WT) and five deletion mutants: membrane

bound cytochrome (ΔmtrC/ΔomcA), transmembrane pili (ΔpilM-Q, ΔmshH-Q, and ΔpilM-

Q/ΔmshH-Q) and flagella (Δflg). We demonstrate that the formal potentials of mediated and

direct electron transfer sites of the derived biofilms can be gained from CVs of the respective

biofilms recorded at bioelectrocatlytic (i.e. turnover) and lactate depleted (i.e. nonturnover)

conditions. As the biofilms possess only a limited bioelectrocatalytic activity, an advanced data

processing procedure, using the open-source software SOAS, was applied. The obtained results

indicate that S. oneidensis mutants used in this study are able to bypass hindered direct electron

transfer by alternative redox proteins as well as self-mediated pathways.

Figure: How does Shewanella transfer its electrons to solid acceptors? Using cyclic

voltammetry direct and mediated electron transfer of S. oneidensis MR-1 and related mutants

were investigated. The subsequent analysis, based on an elaborate open source software data -

processing, indicates a correlation of the maximum current density (x-axes of the graph) of the

respective mutant and its mediated electron-transfer ability (respective CV- peak height on the

y-axes).

Page 48: PhD Thesis Alessandro Carmona2012

-27-

It has been shown for anodic biofilms in MFCs that the microorganisms therein can be

influenced by the applied electrode potential. In chapter 3, we studied the influence of the

applied electrode potential on the anodic current production of Shewanella putrefaciens NCTC

10695. Furthermore, we used cyclic voltammetry (CV) and confocal laser scanning microscopy

(CLSM) to investigate the microbial electron transfer and biofilm formation. It is shown that the

chronoamperometric current density is increasing with increasing electrode potential from 3 µA

cm-2

at -0.1 V up to ~12 µA cm-2

at +0.4 V (vs. Ag/ AgCl), which is accompanied by an

increasing amount of biomass deposited on the electrode. By means of cyclic voltammetry we

demonstrate that direct electron transfer (DET) is dominating and the planktonic cells play only

a minor role.

Figure: Is the current generation, jmax, a function of the applied electrode potential?

Representative chronoamperometric fed-batch cycles of S. putrefaciens at graphite electrodes;

applied potentials: -0.1, 0, +0.1, +0.2, +0.3 and +0.4 V vs. Ag/AgCl; CV measurements during

turn-over (A) and non turn-over (B) conditions respectively.

0 1 2 3 4 5 6 7 8

-2

0

2

4

6

8

10

12

14

16

+0.4

+0.3

+0.2

+0.1

0.0

-0.1

AAA

j ma

x/

A c

m-2

time/ days

BBB

Page 49: PhD Thesis Alessandro Carmona2012

-28-

Crucial for the functioning of bioelectrochemical systems (such as MFCs and MECs) is the

complex protein architecture responsible for the electron transfer (ET) across the

bacteria/electrode interface. The ET pathway involves several multiheme redox proteins denoted

as outer membrane cytochromes (OMCs). In chapter 4 these OMCs were studied by a

combination of surface-enhanced resonance Raman scattering (SERRS) spectroscopy and

electrochemistry. The experiments presented in chapter 4 were performed on microbial biofilms

of S. putrefaciens. These have shown that OMCs do not contribute significantly to the

heterogeneous ET across bacteria/electrode interface. These studies have been performed on

biofilms grown on nanostructured Ag electrodes at the poised potential of +50 mV (vs.

Ag/AgCl). Although these conditions allow the formation of a biofilm on the Ag electrode, they

may have a negative impact on the amount of OMCs expressed by the bacteria (see chapter 3).

In fact, optimal biofilm growth requires pore positive potentials. However, these conditions

cannot be met by the Ag substrate, which undergoes oxidation at potentials higher than +150

mV (vs. Ag/AgCl).

Figure: Electrochemical measurements (A) performed in combination with SERRS (B)

allowed to control and monitor the activity of the microbial biofilm. This chapter aims at

providing the first spectroelectrochemical characterization of microbial biofilms of a strain of

the Shewanellaceae family by probing (i) structural information about the OMCs, (ii) the

participation of the OMCs to the ET, and (iii) the influence of soluble redox mediators

competing with OMCs. The experiments presented here contributed to elucidate the

function/structure relationship of OMCs in living cells, providing unique insight into the ET

across the bacteria/electrode interface. The development of novel analytical strategies to

overcome this limitation is presently under evaluation in our groups.

Page 50: PhD Thesis Alessandro Carmona2012

-29-

In chapter 5 we exploited electroactive bacteria in bioelectrochemical systems like MFCs that

promise a great potential in the context of sustainable energy supply and handling. A major

challenge in this context is to increase the performance of such systems, a necessity for a future

success of this new technology. During the past decade the average current densities of biofilm

anodes have already increased significantly from milliampere per square metre level to between

7 and 10 A m-2

. In this study it is demonstrated that by using three-dimensional carbon fiber

electrodes prepared by electrospinning and solution blowing the bioelectrocatalytic anode

current density reaches values of up to 30 A m-2

, which represents the so far the highest reported

values for electroactive microbial biofilms.

Figure: How do electroactive bacteria benefit from 3D materials? A) Biocatalytic current

generation at a 3D porous carbon fiber produced by gas-assisted electrospinning (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 vs. Ag/ AgCl. B) Scanning electron

microscopic image of an electroactive biofilm grown at a high porosity three-dimensional

structure carbon felt GES-CFM. The excellent bioelectrocatalytic performance of this material is

attributed to a structure that provides a habitat for the growth of electroactive bacteria up to a

maximum density supplemented by efficient substrate supply through the open pore structure.

The interconnections between the individual fibers of the nonwoven allow the formation of

cross-linked three-dimensional biofilms that benefit from an optimum electron transfer and

conduction.

Page 51: PhD Thesis Alessandro Carmona2012

-30-

In chapter 6 layered carbon fiber mats have been prepared by layer-by-layer (LBL)

electrospinning of polyacrylonitrile onto thin natural cellulose paper and subsequent

carbonization. The layered carbon fiber mat (CFM) has been proven to be a promising BES

anode material for MFCs and MECs, allowing high density layered biofilm propagation and

thus high bioelectrocatalytic anodic current density. Thick and continuous layered biofilms were

grown on these layered-carbon nanofiber mats and generated high current densities from waste

water derived biofilms. This investigation also revealed that, if the gap between the layers

within the layered-carbon nanofiber mats can be further increased in order to allow ideal

nutrient availability, thick layered biofilms might grow in every layer of the entire layered-CFM

and much larger current densities would be obtained. In summary, the cellulose-based carbon

fiber mat provides a low cost and highly efficient material for bioelectrocatalytic anodes in

microbial fuel cells.

Figure: Layered architecture of anode materials promotes the growth of electroactive

biofilms. A) Chronoamperometric (0.2 V vs. Ag/ AgCl) Biocatalytic current generation curves

from waste water derived biofilms of carbon fiber mats in a half-cell experiment; and B) SEM

image of a thick and continuous layered biofilm grown in the layered-CFM. In summary, here it

has been showed that small fiber diameter and proper pore size combined with sufficient three -

dimensionality- are essential features for the growth of high performance thick and continuous

biofilms.

Page 52: PhD Thesis Alessandro Carmona2012

-31-

It has been assumed that the treatment of wastewater could be one of the most appealing

applications for some BESs such as MFCs. Therefore, in order to make MFCs a successful

technology in wastewater treatment, researchers have to pay special attention to the

environmental and external factors that influence the biofilm. In chapter 7 the pH-value played a

crucial role for the development and current production of anodic microbial electroactive

biofilms. It was demonstrated that only a narrow pH-window, ranging from pH 6 to pH 9, was

suitable for growth and operation of biofilms derived from pH-neutral wastewater. Any stronger

deviation from pH neutral conditions led to a substantial decrease in the biofilm performance.

Thus, average current densities of 151 µA cm-2

, 821 µA cm-2

and 730 µA cm-2

were measured

for anode biofilms grown and operated at pH 6, 7 and 9 respectively. The microbial diversity of

the anode chamber community during the biofilm selection process was studied using the low

cost method flow-cytometry. Thereby, it was demonstrated that the pH value as well as the

microbial inocula had an impact on the resulting anode community structure. As shown by

cyclic voltammetry the electron transfer thermodynamics of the biofilms was strongly

depending on the solution’s pH-value.

Figure: Effect of pH in the performance of potentiostatic fed-batch electro-active biofilms

derived from primary wastewater. The more the pH-value during biofilm formation and

operation deviates from the pH of the bacterial source (pH neutral wastewater), the lower and

less efficient its bioelectrocatalytic activity becomes.

Page 53: PhD Thesis Alessandro Carmona2012

-32-

In chapter 8 the investigation of environmental parameters that affect the formation and

performance of anodic electroactive biofilms in MFCs were studied. In order to exclude the

influence of operational variables and to investigate only the effect of an individual microbial

inoculum source and an individual substrate, the experiments were conducted with half-cell set-

ups under potentiostatic control. A significant difference in current generation was observed for

all bioelectrochemical set-ups. Acetate-fed-reactor with primary wastewater inoculum showed

the highest current density (558 ± 27 μA cm-2

), followed by lactate-fed-reactor with primary

waste water inoculum (460 ± 54 μA cm-2

). The high performance with primary wastewater for

the formation of bioelectroactive biofilms demonstrated its ability as efficient microbial

inoculum source. Cyclic voltammograms (CVs) of all biofilms indicated the different electro-

chemical behaviour with both substrates. Maturity of biofilms was confirmed from a constant

maximum of current density production and a non-changing CV shape after several semi-batch

cycles (only for biofilms enriched from primary wastewater). For turnover CVs of biofilms

enriched from primary wastewater and both substrates the formal potential of the active site was

about -260 mV vs. Ag/ AgCl (see Figure). This clearly indicates that the used inocula

considerably influenced the enrichment of electrochemically active bacteria. For non-turnover

CVs, the electrochemical characterization of the biofilms reveals a strong similarity to

Geobacter sulfurreducens biofilms, which may indicate a dominating role of this bacterium in

the biofilms enriched from primary wastewater as source of inoculum.

Figure: Influence of the inoculum and substrate on the formation of electro-active

biofilms. Here exemplary CVs are shown for biofilms derived from primary wastewater set-ups,

the only experimental set-ups with a constant CV shape after the third semi-batch cycle.

Page 54: PhD Thesis Alessandro Carmona2012

-33-

CHAPTER II

2 Cyclic voltammetric analysis of the electron transfer of

Shewanella oneidensis MR-1 and nanofilament and

cytochrome knock-out mutants

2.1 Introduction

Electrochemically active bacteria (EAB) can transfer electrons to solid terminal electron

acceptors such as Fe(III), Mn(III), Cr(VI), and even to carbon electrodes in microbial fuel cells

(MFCs) (Chang, et al., 2006). These bacteria not only play a key role in nature's oxidation–

reduction cycles (Nielsen, et al., 2010) but also are the key component of microbial

bioelectrochemical systems (BES) (Rabaey, et al., 2010) (for an example on a BES see Fig. S9-1

in Supplementary information for Chapter II). Thus, the elucidation of the different microbial

electron transfer pathways is of fundamental interest as well as technological relevance.

Up to now, several classes of extracellular electron transfer mechanisms have been elucidated

for a wide range of microorganisms (Logan, 2009, Schröder, 2007). In principle, direct electron

transfer (DET) and mediated electron transfer (MET) can be distinguished. Whereas DET relies

on the physical contact of the redox active bacterial moiety, including redox proteins like

cytochromes (Busalmen, et al., 2008, Wigginton, et al., 2007) or bacterial nanowires (Gorby, et

al., 2006, Reguera, et al., 2005), with the solid terminal electron acceptor (e.g. iron (III) mineral

or anode of a BES), no such direct contact is necessary for MET. With MET, a dissolved

chemical compound that can serve as electron shuttle, i.e. mediator, facilitates the electron

transfer (Marsili, et al., 2008a, Rabaey, et al., 2005).

A wealth of different microorganisms, including single strain cultures as well as mixed

consortia, have been shown to be electrochemically active (Logan, 2009, Rabaey, et al., 2007)

and thus the analysis of the electron transfer pathways is actively being investigated. The

predominant model organisms studied are from the families Geobacteraceae (Lovley, 2008b)

and Shewanellaceae (Nealson and Scott, 2006). Whereas the electrochemical analysis of the

DET for Geobacter is well established (Fricke, et al., 2008, Marsili, et al., 2010, Srikanth, et al.,

2008), the latter microbe has been shown to possess more complex bioelectrochemical behavior

and the ability to generate sustained power in MFCs, even under constant exposure to dissolved

Page 55: PhD Thesis Alessandro Carmona2012

-34-

oxygen. In a recent study by Baron et al., cyclic voltammetry (CV) was used to demonstrate that

there are two redox active centres in adsorbed cells of Shewanella oneidensis; one responsible

for DET and one responsible for MET (Baron, et al., 2009). Furthermore, the authors showed

that the addition of exogenous flavins enhances mediated electron transfer. Recently, S.

oneidensis wild type and several electron transfer relevant mutants have been shown to differ

significantly concerning their bioelectrochemical activity (Bouhenni, et al., 2010).

Therefore, the aim of this study was to investigate the electron transfer properties of the

respective mutants within biofilms which were grown on an active electrode (i.e. in situ) using

cyclic voltammetry. A summary on electron transfer pathways for S. oneidensis and the

respective mutants used within this study are illustrated in the following section.

2.1.1 Extracellular electron transfer mechanisms of S. oneidensis MR-1 wild type and

mutants

The literature describing electron transfer mechanismsof S. oneidensis provides a complex

picture (see e.g. (Beliaev, et al., 2005, Bouhenni, et al., 2010, Gorby, et al., 2006, Hartshorne, et

al., 2007)) and is summarized in Fig. 2-1.

2.1.1.1 Direct electron transfer (DET)

For the facultative anaerobe S. oneidensis MR-1, there are two decahemes c-type cytochromes

(MtrC and OmcA) which are exposed on the outer cell surface and are assumed to be key

proteins for DET (Eggleston, et al., 2008, Firer-Sherwood, et al., 2008b, Fredrickson, et al.,

2008, Meitl, et al., 2009, Xiong, et al., 2006), yet are also reported to possess only limited rate

constants for electron transfer compared to mediator molecules (Baron, et al., 2009, Peng, et al.,

2010b).

Furthermore, as depicted in Fig. 2-1A these proteins can play a role in the mediated electron

transfer (MET) as the mediator reduction may take place on the outer cell surface. According to

the present models, this cytochrome facilitated DET and MET can be performed by the wild

type (WT) and all mutants used within this study, except ΔmtrC/ΔomcA due to a lack of outer

surface cytochromes. In addition to the cytochrome based electron transfer, pili outer surface

structures may play a role in extracellular electron transport. As shown in Fig. 2-1B and C, these

pili are believed to contribute to the microbial DET and MET.

Page 56: PhD Thesis Alessandro Carmona2012

-35-

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.

Page 57: PhD Thesis Alessandro Carmona2012

-36-

Furthermore, these pili are proposed to play a key role in the microbial cell attachment and thus

biofilm formation. Previous studies find that the three pili biogenesis knock-out mutants used

within this study (ΔpilM-Q, ΔmshH-Q and ΔpilM-Q/ΔmshH-Q) as well as the flagellin knock-

out mutant (Δflg) possess reduced biofilm forming ability (see Fig. 2-1D) (Bouhenni, et al.,

2010, Thormann, et al., 2004).

2.1.1.2 Mediated electron transfer (MET)

Several strains of the genus Shewanella can biosynthesize mediators to facilitate electron

transfer to the terminal electron acceptor. These mediators have been shown to be based on

flavins (e.g. (Biffinger, et al., 2010, Bouhenni, et al., 2010, Marsili, et al., 2008a, von Canstein,

et al., 2008)). For S. oneidensis, flavinmononucleotide (FMN) and riboflavin have been shown

to be redox shuttles in aerobic as well as anaerobic conditions (Velasquez-Orta, et al., 2010, von

Canstein, et al., 2008). In principle, a mediator molecule can be reduced in the inner cell or outer

cell membrane surface structures (Fig. 2-1A to C).

2.2 Materials and methods

2.2.1 General conditions

All chemicals were of analytical or biochemical grade and were purchased from Sigma-Aldrich

and Merck. If not stated otherwise, all potentials provided in this article refer to the Ag/AgCl

reference electrode (sat. KCl, 0.195 V vs. SHE). All microbial experiments were performed

under strictly sterile conditions.

2.2.2 Cell cultures and media

Sterilized LB Broth (Ringeisen, et al., 2006), and minimal media (Biffinger, et al., 2008) for

liquid cultures and LB/agar (Merck KGaA, Germany) plates were used for culture maintenance.

Single colonies of S. oneidensis MR-1 wild type and mutants (ΔmtrC/ΔomcA, ΔpilM-Q,

ΔmshH-Q, ΔpilM-Q/ΔmshH-Q and Δflg; obtained as reported elsewhere (Bouhenni, et al.,

2010)) were transferred to 15 mL of LB broth and incubated aerobically at room temperature

while shaking at 100 rpm (Universal shaker SM 30 A, Edmund Bühler GmbH, Germany) for 48

h. Afterwards, 15 mL of minimal media were added. Then 5 mL was used for subcultures in

growth medium (18 mmol/L Sodium lactate, PIPES Buffer 15.1 g/L; NaOH 3.0 g/L; NH4Cl 1.5

g/L; KCl 0.1 g/L; NaH2PO4∙H2O 0.6 g/L; NaCl 5.8 g/L; Mineral solution 10 mL/L (Atlas,

1993); Vitamin solution 10 g/L (Atlas, 1993); Amino acid solution 10 g/L (Bretschger, et al.,

2007)).

Page 58: PhD Thesis Alessandro Carmona2012

-37-

2.2.3 Bioelectrochemical experiments

The bioelectrochemical experiments were carried out under potentiostatic control

(Potentiostat/Galvanostat VMP3, BioLogic Science Instruments, France) utilizing a three-

electrode arrangement, with a carbon rod working electrode (2.5 cm height×1.0 cm diameter,

CP-Graphite GmbH, Germany), a Ag/AgCl reference electrode (sat. KCl, Sensortechnik

Meinsberg, Germany) and a carbon rod (4.5 cm high×1.0 cm diameter) as counter electrode -

shielded by a 117 Nafion membrane. Sealed vessels (250 mL) served as the electrochemical cell

hosting the three-electrode arrangement (see Fig. S9-2 in Supplementary information). The

biofilm growth was performed in semi-batch chronoamperametric experiments at +0.2 V with

regular media replacement (addition of 200 mL fresh media solution to the 50 mL in the cell).

During these potentiostatic biofilm growth experiments aerobic conditions were assured

(Rosenbaum, et al., 2010a, von Canstein, et al., 2008) by pumping filtered air in the cells using

one fish pump (Elite air pump 799, Rolf C. Hagen Corp., Mansfield, MA. 02048, USA) per six

cells. Fresh electron donor and nutrients were supplied about every 24 h by removing 200 mL of

culture and replacing with 200 mL of fresh growth media.

Cyclic voltammetry (CV) was recorded during turnover conditions (TC), i.e. at the

bioelectrocatalytic substrate consumption, and during non-turnover (NTC), i.e. substrate

deprived, conditions at a scan rate of 1 mV s−1

. All CV experiments were performed under

anoxic conditions which were achieved before every experiment by bubbling nitrogen for 15

min in the solution. The headspace of the solution was also sparged with nitrogen during the

CV-measurement.

2.2.4 Data processing

Chronoamperometric maximum current densities (calculated per projected electrode surface

area) during at least 18 semi-batch cycles for established microbial biofilms for 3 independent

biofilm replicates were analyzed (see Figure S9-3 in Supplementary information). The standard

deviations are presented in Table 2-1. For in-depth data analyses of the cyclic voltammograms,

the open-source software SOAS (Fourmond, et al., 2009) was used for baseline (capacitive

current) correction for non-turnover conditions. Furthermore by using this software, the non-

turnover data was subtracted from the respective turnover data, and first derivatives were

calculated. Here all data are based on experiments during at least 18 semi-batch cycles of the 3

independent biofilm replicates, and the standard deviations are presented in Fig. 2-4.

Page 59: PhD Thesis Alessandro Carmona2012

-38-

2.3 Results and discussion

2.3.1 Bioelectrochemical current production

Table 2-1 summarizes the maximum chronoamperometric current densities, derived from semi-

batch chronoamperometric experiments at 0.2 V vs. Ag/AgCl, for biofilms grown under aerobic

conditions of the S. oneidensis WT and the five mutant biofilms under investigation using

18mmoL/L lactate as the electron donor.

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.

Strain Mutant description

jmax / µA cm-2

This

worka

Ref.

Ab

Ref.

Bc

Ref.

Cd

Ref.

De

∆flg Flagella deletion mutant 9.5 ± 2.0 8 - - -

Wild Type S. oneidensis MR-1 wild-type,

ATCC 700550 7.9 ± 1.5 5 13 3.4 6

ΔpilM-Q Type IV pilus deletion mutant 7.7 ± 3.7 7.6 - - -

∆mtrc/

∆omcA

Outer membrane decaheme

c-type cytochromes MtrC and

OmcA deletion mutant

4.3 ± 0.8 0.6 2 0.7 <1

ΔmshH-Q Mannose-sensitive hemagglutinin

pilus deletion mutant 3.6 ± 1.9 3.2 - - -

ΔpilM-Q/

ΔmshH-Q

Type IV pilus and mannose-sensitive

hemagglutinin pilus deletion mutant 1.4 ± 1.6 2.2 - - -

aAverage data from chronoamperometric experiments at 0.2 V vs. Ag/AgCl calculated as

described in Section 2.4 and its respective standard deviation.bMFC experiments at an external

resistance of 100 kΩ after 200 h. c

MFC experiments at an external resistance of 10 Ω. d

MFC

experiments at an external resistance of 10 Ω. eChronoamperometric experiments at 0.043 V vs.

Ag/AgCl. Ref. A: (Bouhenni, et al., 2010); Ref. B: (Bretschger, et al., 2007); Ref. C: (Gorby, et

al., 2006); Ref. D: (Coursolle, et al., 2010).

All chronoamperometric biofilm growth experiments were made under oxygen exposure as

previous reports have shown that S. oneidensis WT preferentially forms thicker biofilms under

air-exposed conditions (Biffinger, et al., 2009). The growth pattern suits well with previous

Page 60: PhD Thesis Alessandro Carmona2012

-39-

reports at other electrode potentials (e.g., 0.0 V vs. SCE (Peng, et al., 2010a) or 0.0 to 0.5 V vs.

Ag/AgCl (Peng, et al., 2010a)) and microbial activity was maintained for more than 18 days. For

comparison, Table 2-1 shows the current densities of the previous study on the respective

mutants under non-agitated air-exposed conditions in a microbial fuel cell study (Bouhenni, et

al., 2010, Bretschger, et al., 2007, Coursolle, et al., 2010, Gorby, et al., 2006). When further

comparing the WT current densities, the achieved maximum current density is well within the

expected range (Rosenbaum, et al., 2010a) - see Table S9-1 in Supplementary Information. The

focus of our analysis was the respective electron transfer mechanisms using cyclic voltammetry

and not maximizing current output as would be typical for MFC research.

2.3.2 Cyclic voltammetric analysis and data processing

In order to analyze the extracellular electron transfer mechanisms, cyclic voltammetry was

performed for all microbial biofilms/ suspensions during the growth cycles at maximum current

density (i.e. turnover) and at substrate depletion (i.e. non-turnover). Representative CVs for both

conditions are shown in Fig. 2-2A and B (nonturnover) and Fig. 2-3A and B (turnover).

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.

Page 61: PhD Thesis Alessandro Carmona2012

-40-

Noteworthy, to avoid the oxygen disturbance on the CV measurements all experiments were

performed under strictly anoxic conditions. Furthermore, it has to be mentioned that initial

experiments where all bacteria were grown under anaerobic conditions did not yield sustainable

chronoamperometric and CV measurements.

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

.

As the CVs for both conditions do not show easily interpretable voltammetry (as it is the case

for e.g. Geobacter), the analysis of potential and actual electron transfer sites is not

straightforward (Fricke, et al., 2008). This behaviour can be attributed to the fact that the CV

current densities for turnover and non-turnover conditions vary less than one order of

magnitude, which indicates a rather small catalytic effect and hence low bioelectrocatalytic

activity of S. oneidensis. Noteworthy, the comparably high background current (that is due to

non-catalytically active, yet redox active microbial moieties and the exo-polysaccharide matrix)

in relation to the bioelectrocatalytic current densities might point towards i) a lower abundance

of redox centres per biomass and/ or ii) a lower turnover of the individual redox centre.

However, this question needs further investigation.

Thus, alternative, elaborate procedures for the CV analysis had to be followed. By using SOAS

(Fourmond, et al., 2009) baseline, i.e. capacitive current, corrected CVs for non-turnover

conditions could be generated - as shown in Fig. 2-2C and D. From these plots it can be

concluded that all studied bacterial strains use two different electron transfer pathways,

possessing formal potentials, Ef, of about -330 ± 45 mV (redox system I) and −70 ± 17 mV

(redox-system II), respectively.

Page 62: PhD Thesis Alessandro Carmona2012

-41-

Redox system I can be attributed to mediated electron transfer as its potential is in the range for

that found for soluble electron shuttles of Shewanellae in other studies (Biffinger, et al., 2008,

Marsili, et al., 2008a, Peng, et al., 2010b). However, the differing formal potential may be

attributed to the experimental conditions, e.g. free vs. biofilm-bound mediator and electrode

material, and at the electrode surface. Here a pH-decrease of the anodic reaction medium as well

as a pH-gradient near to the electrode surface (Torres, et al., 2008) will lead to a positive shift of

the formal potentials. In our potentiostatic set-ups, using shielded counter electrodes, we found

pH-values as low as pH 6 at the end of the growth cycle in the anodic compartment. This

influence of pH-microenvironment accounts as well for the membrane bound electron transfer

proteins, for which the redox-system II may be ascribed to a DET mechanism (Kim, et al.,

1999b).

The formal potentials of the surface proteins OmcA and MtrC varies within the literature. For

instance for a purified MtrC-protein, Ef-MtrC was found to be −270 mV vs. Ag/AgCl in pH 7

buffer solutions using basal plane graphite electrodes (Hartshorne, et al., 2007). Using Fe2O3

electrodes, Ef-OmcA was measured to be −210 mV when suspended in solution or −160 to −130

mV when adsorbed on the electrode surface (Eggleston, et al., 2008).

When studying whole cell suspensions of respective single knock-out mutants on haematite

electrodes, Ef-OmcA = −159 mV was identified (Meitl, et al., 2009). By measuring attached cells

on graphitic carbon electrodes, both formal potentials were estimated to be about −200 mV

(Baron, et al., 2009). Hence, these cytochromes possess a broad potential window of about 300

mV (Firer-Sherwood, et al., 2008b) and their actual formal potential seems strongly dependent

upon their microenvironment, and also here the pH-value direct at the electrode surface plays a

decisive role.

Furthermore, the potentially pilin-facilitated electron transfer mechanism was analyzed using the

Δflg, ΔpilM-Q, ΔmshH-Q, and ΔpilM-Q/ΔmsH-Q knock-out mutants. No discrimination from

the cytochrome-related signals was possible and thus no individual formal potentials of the pili-

related electron transfer could be identified, especially as no studies on the extracted

transmembrane moieties (ΔpilM-Q and ΔmshH-Q) are available. However, our data suggest that

the respective pili-related formal potentials are close to that of OmcA and MtrC, as all these

DET mechanisms are ultimately dependent on the same intracellular redox chains. This

assumption might furthermore explain why a DET signal was detected for ΔmtrC/ΔomcA that

may then be caused by the pili-related DET).

Page 63: PhD Thesis Alessandro Carmona2012

-42-

In order to elucidate the reasons for the deviating bioelectrocatalytic activities of the S.

oneidensis WT and mutants - that could not be explained on differing numbers of active cells, an

elaborate analysis of the CV data for turnover (presented below) and non-turnover conditions

was performed. For non-turnover conditions, we analyzed the dependence of the maximum

bioelectrocatalytic activity on the peak properties of the baseline corrected CVs (Fig. 2-2C and

D). Here it was found that the peak height of the oxidation peak of the redoxsystem I, jpeak, was

a function of the chronoamperometric maximum current density, jmax (Table 2-1). This

dependency (Fig. 2-4) indicates that the concentration of the mediator molecule, which can be

assumed to correlate linearly with the respective height of the oxidation peak of redox-system I,

is required for the maximum current generation.

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.

Interestingly, this finding points towards an only inferior role of the DET mechanism for WT

and all mutants, which may sound logical when the low electron transfer rate constants of the

outer surface proteins mtrC and omcA are considered (Peng, et al., 2010b). Interestingly, for all

other peak areas and heights, such dependencies could not be identified (data not shown; finding

no correlation of jmax to the respective reduction peak of redox-system I has to be attributed the

Page 64: PhD Thesis Alessandro Carmona2012

-43-

limited CV-range, i.e. close vicinity of the signal to the vertex potential of the CV.). This is

contrary to our initial expectations that DET-related CV signal may be reduced for pilin-deletion

mutants. Yet, this finding might be explained as discussed below.

The analysis of the CVs for turnover conditions was not straightforward, as the calculation of

the derivatives did not yield a clear picture on the electron transfer sites. Therefore, we

performed the following analysis: The CVs for non-turnover conditions were subtracted from

the CVs for turnover conditions (Fourmond, et al., 2009), resulting in curves depicting the net-

catalytic current (Bard, et al., 2008). As Fig. 2-5 shows for the WT of S. oneidensis, two

catalytic waves could be identified. As the first half wave potential (approx. −300 mV) is very

similar to the formal potential of redox-system I - and thus found for flavin based mediators in

literature (Marsili, et al., 2008a) - it can clearly be attributed to MET. The ascription of the

second catalytic wave is more complex, as even its onset potential is more negative than the

formal potential of redox system II that was identified from non-turnover CVs (see above).

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).

Additionally, as can be clearly seen from Fig. 4-5, a half wave potential cannot be identified.

The latter finding may be attributed to several phenomena: First, the evolving proton gradient

Page 65: PhD Thesis Alessandro Carmona2012

-44-

within the biofilm (Torres, et al., 2008) may lead to a (continuous) shift of the potential of the

electron transfer site. This finding is supported by recent studies investigating the pH

dependence of mixed culture biofilms (Patil, et al., 2011). Second, this result could be due to

phenomena that are well known from enzyme electrochemistry. Here analogue shaped catalytic

curves are ascribed to i) an inhomogenous coupling of the redox molecules to the electrode

surface and/or ii) slow electron transfer kinetics in relation to the substrate conversion kinetics

(Vincent, et al., 2007). Both factors are likely to play a role for whole cell biofilms. Noteworthy,

after smoothing and interpolation of the (non-turnover corrected) turnover CV curves, suitable

derivatives can be observed. For our measurements (Fig. S9-4 – Fig. S9-7 in the Supplementary

Information for Chapter 2) the maxima of these derivatives corresponds very well with the

formal potentials identified above.

Both catalytic waves can be found in all mutants, which indicate that in none of the microbial

cells was the DET completely inhibited, which is in line with our findings for non-turnover

conditions (see above). This result redox-signal when attached biofilms (Baron, et al., 2009) or

cell suspensions (Meitl, et al., 2009) were studied. However, when taking into account that S.

oneidensis possesses more than 42 (Meyer, et al., 2004) different cytochromes, of which 80%

are localized to the outer membrane (Heidelberg, et al., 2002), this finding might further support

the hypothesis that when one redox-protein is knocked out, other “bypass” molecules (including

other OMCs and/or pili) are used. In this context, two additional molecular biological studies

should be mentioned that illustrate the high versatility and complexity of S. oneidensis energy

metabolism. Kolker and co-workers revealed by a transcriptomics and proteomics based

analyses of 538 hypothetical genes, representing one third of the predicted number of proteins of

S. oneidensis MR-1 (Kolker, et al., 2005), that the respiratory versatility of this microorganism

may be explained not only by the high number of c-type cytochromes but also the existence of

numerous further, specialized genes that are directly related to the microbial energy conversion.

Beliaev et al. (Beliaev, et al., 2005), examined the globalmRNApatterns of S. oneidensis by

exposing the bacteria to different metal and non-metal electron acceptors. They have found that

only one of the 42 predicted c-type cytochromes (SO3300) displayed significantly elevated

transcript levels across all metal-reducing conditions. It has therefore been assumed that this

flavocytochrome c possessing a subunit with 4 hemes, participates significantly in the energy

metabolism and specifically possesses an outer surface electron transport role. Other mRNA-

levels, most prominently of MtrC and MtrA, were also slightly decreased in the presence of

metals.

Page 66: PhD Thesis Alessandro Carmona2012

-45-

As an opportunity to (roughly) estimate the contribution of both electron transfer pathways (i.e.

DET and MET) to the bioelectrocatalytic activity of a respective microorganism, we propose the

following procedure (Fig. 2-5). The signal height of each catalytic wave is estimated at suitable

fixed potentials, where an almost full catalytic activity can be expected (here −0.2 V vs.

Ag/AgCl and 0.1 V vs. Ag/AgCl) and thus the share of each catalytic centre to the overall

maximum current can be estimated. The results of this analysis are summarized in Table 2-2.

The relative current share of redox-system I (Δi−0.2) is increased and consequently the share of

redox-system II (Δi+0.1) is decreased for all mutants (except ΔpilM-Q) in comparison to the wild

type. This finding indicates an increased contribution of mediated electron transfer when pili,

outer surface cytochromes or flagella are not available as DET pathways. However, the

differences, especially taking into account the standard deviations, are only minor for most of

the mutants — most pronounced with a MET share of 70% for motility inhibited mutant Δflg.

This finding corresponds well with the data presented in Fig. 2-4 and is an additional piece of

the complex extracellular electron transfer puzzle for Shewanella.

Table 2-2 Result of the CV subtraction analysis (details in Fig. 5 and the text).

Strain Relative share of the current/%

Redox-system I Redox-system II

ΔpilM-Q/ΔmshH-Q 57 ± 19 42 ± 16

ΔpilM-Q 48 ± 24 51 ± 43

Wild-type 52 ± 27 47 ± 16

ΔmshH-Q 61 ± 26 38 ± 14

Δflg 71 ± 19 28 ± 14

ΔmtrC/ΔomcA 62 ± 44 37 ± 17

Page 67: PhD Thesis Alessandro Carmona2012

-46-

2.4 Conclusions

In this study we exploited cyclic voltammetry as a tool for the in situ study of S. oneidensis wild

type and mutant biofilms grown at electrode surfaces. Since the catalytic activity of these

microorganisms was found to be limited (as reflected by only low current densities) the analysis

of the electron pathways is not as straightforward as for bacteria that do not possess the ability to

self-mediate extracellular electron transfer (Fricke, et al., 2008). In order to analyze the CV data,

an elaborated data processing was performed. By this data analysis, the formal potentials of the

direct and mediated electron transfer were identified and the share of each electron transfer

pathway to the overall current production could be estimated. However, it was not possible to

elucidate thermodynamic/ mechanistic differences in the direct electron transfer for respective

knock-out mutants. Furthermore our results indicate that mutants possessing knock-outs for

potential DET-related proteins bypass this deficiency by alternative DET redox-carriers and

self-mediated pathways. Here, more sensitive electrochemical (e.g. square wave voltammetry),

spectroscopic and related techniques in combination with molecular biological approaches (e.g.

transcriptomics) need to be exploited in future research.

Page 68: PhD Thesis Alessandro Carmona2012

-47-

CHAPTER III CHAPTER III

3 Study of Shewanella putrefaciens biofilms grown at

different applied potentials using cyclic voltammetry and

confocal laser scanning microscopy

3.1 Introduction

Although, the ability of microorganisms to create an electrochemical potential was discovered

more than 100 years ago (Potter, 1911), it was mainly during the last decade that this field of

research was developing most dramatically (Schröder, 2011), as it was found that the ability to

transfer electrons to extracellular electron acceptors is naturally occurring in several microbial

species. This interest thereby is triggered by fundamental inquisitiveness (Hernandez and

Newman, 2001, Schröder, 2007, Watanabe, et al., 2009) as well as by the development of

seminal sustainable technologies based on microbial extracellular electron transfer - also known

as microbial bioelectrochemical systems (BES) (Rabaey, 2010, Rabaey and Rozendal, 2010).

Up to now, several microorganisms have been studied in order to elucidate their specific

extracellular electron transfer mechanisms, including Lactococcus lactis (Masuda, et al., 2010),

Saccharomyces cerevisiae (Ducommun, et al., 2010), Pseudomonas sp. CMR12a (Pham, et al.,

2008), Hansenula anomala (Prasad, et al., 2007), Proteus vulgaris (Rawson, et al., 2011) and

Lyngbya sp. and Nostoc sp. (Pisciotta, et al., 2011). Here, the two families of Gram-negative

bacteria Shewanellaceae (Myers and Nealson, 1988, Nealson and Scott, 2006) and

Geobacteraceae (Caccavo, et al., 1994, Lovley, 2008b, Malvankar, et al., 2011, Richter, et al.,

2009) are the most widespread model organisms. Thereby, different electron transfer strategies

have been commonly described among the various species, which are summarized as follows: i)

direct electron transfer (DET) via membrane-bound redox-enzymes (Inoue, et al., 2011, Millo,

et al., 2011, Strycharz, et al., 2011) or via bacterial nanowires (El-Naggar, et al., 2010, Gorby, et

al., 2006, Malvankar, et al., 2011, Reguera, et al., 2005) and ii) mediated electron transfer

(MET) via redox shuttle substances, e.g. (Jiang, et al., 2010, Marsili, et al., 2008a), usually

secondary microbial metabolites.

Page 69: PhD Thesis Alessandro Carmona2012

-48-

For the family of Shewanellaceae – all being considered to be facultative anaerobes - several

members have been studied on their extra-cellular electron transfer behavior. Most prominently

S. oneidensis MR-1 (Baron, et al., 2009, Meitl, et al., 2009, Okamoto, et al., 2011, Sun, et al.,

2010), but also S. oneidensis MR-4 (Marsili, et al., 2008a), S. putrefaciens W3-18-1

(Bretschger, et al., 2010a), S. putrefaciens IR-1 (Kim, et al., 1999b, Kim, et al., 1999d, Kim, et

al., 2002), S. putrefaciens SR-21 (Kim, et al., 2002), S. loihica PV-4 (Bretschger, et al., 2010a,

Nakamura, et al., 2009a, Nakamura, et al., 2009b, Okamoto, et al., 2009, Zhao, et al., 2010b), S.

decolorationis NTOU1 (Li, et al., 2010, Li, et al., 2009a), S. japonica KMM 3299 (Biffinger, et

al., 2010), S. frigidimarina NCIMB400 (Pankhurst, et al., 2006, Turner, et al., 1999) and S.

marisflavi EP1 (Huang, et al., 2010). Commonly, it is assumed that the two decaheme c-type

cytochromes MtrC and OmcA, both facing the extracellular environment, play a key role in the

direct electron transfer mechanism (DET) (Eggleston, et al., 2008, Firer-Sherwood, et al.,

2008b, Fredrickson, et al., 2008, Hartshorne, et al., 2007, Meitl, et al., 2009, Xiong, et al.,

2006). Thereby, MtrC and OmcA, are part of a complex transmembrane cytochrome pool

involving more than 40 proteins (Beliaev, et al., 2005).

The MET of Shewanellaceae exploits redox-shuttles including humic substances (Hong, et al.,

2007), melanin (Turick, et al., 2009, Turick, et al., 2002), menaquinone (Lies, et al., 2005) as

well as riboflavin (von Canstein, et al., 2008), flavinmononucleotide (Biffinger, et al., 2010,

Velasquez-Orta, et al., 2010) and their derivatives. Further it is considered to depend on

intracellular electron transfer to the redox-shuttle as well as extracellular electron transfer by

MtrC or OmcA, e.g. (Biffinger, et al., 2010, Bouhenni, et al., 2010, Marsili, et al., 2008a).

Thereby it has been shown that some strains of the Shewanella family (e.g. S. oneidenis MR-1

(Jiao, et al., 2011, Marsili, et al., 2008a), S. loihica PV-4 (Newton, et al., 2009), S. baltica

Os155 and Os195 (von Canstein, et al., 2008), S. frigidimarina NCIMB400 (von Canstein, et al.,

2008) and S. decoloratioans NTOU1 (Li, et al., 2010)) can biosynthesize these mediators under

aerobic as well as anaerobic cultivation. However, the ability to synthesize suitable amounts of

these electron shuttles (especially for anaerobic conditions, where energy for biosynthesis is

limited) is not unequivocally established for all Shewanella species, e.g. (von Canstein, et al.,

2008). In this course it is of crucial importance to consider the exact conditions during biofilm

growth and development (Harnisch and Rabaey, 2012).

Page 70: PhD Thesis Alessandro Carmona2012

-49-

3.1.1 Influence of the electrode potential on electroactive microbial biofilms

It has been shown for anodic mixed culture derived biofilms that the applied electrode potential

determines decisively the bacterial composition, e.g. (Torres, et al., 2009), of the gained biofilm.

However, also pure culture anodic biofilms and the microorganisms therein can be influenced

by the applied electrode potential. Cho and Ellington (Cho and Ellington, 2007) demonstrated

for S. oneidensis MR-1 that during chronoamperometric biofilm growth the lag-period

decreased from 90 days at 0 mV to 5 days at 500 mV (vs. Ag/AgCl), respectively with

increasing electrode potential; whereas the current density was almost constant. Furthermore,

Shewanella oneidensis MR-1 is believed to show some motility towards electrodes and thus the

availability of its cellular appendices (wires, pili and flagella) clearly determines its electron

transfer performance (Carmona-Martínez, et al., 2011). Recently Harris and colleagues (Harris,

et al., 2010) observed an increase in cell swimming speed when whole cells of Shewanella

species (S. oneidensis MR-1, S. amazonensis SB2B and S. putrefaciens CN32) were exposed to

varying electrode potentials. In addition, Liu and colleagues (Liu, et al., 2010a) investigated

microbial extracellular electron transfer activity of S. loihica PV-4 by applying different

electrode potentials (from -0.4 V to 0.2 V vs. Ag/ AgCl). Thereby, they demonstrated a clear

dependence of the activity on the applied electrode potential, with an activity increase for

potentials more positive than -220 mV. Furthermore, Liu et al. (Liu, et al., 2011) have recently

shown on the example of S. oneidenis MR-1 and S. loihica PV-4 that, depending on the

potential of bacteria cultivation, the electron transfer pathways can be switched from DET to

MET and the formal potential of the DET (Liu, et al., 2010a) is also triggered by the redox-

conditions during cultivation.

Within the context of correlating the applied electrode potential (i.e. potential of the microbial

terminal electron acceptor) and the bacterial activity, it was the aim of this study to elucidate the

electrochemical response of the S. putrefaciens NCTC 10695, a strain that is studied in BES for

the first time. For a comparison with other S. putrefaciens see table S10-2. Therefore, the

influence of the applied electrode potential on the electron transfer mechanisms was investigated

using cyclic voltammetry (CV) and biofilm morphology by means of confocal laser scanning

microscopy (CLSM). CLSM has recently been applied for the study of electroactive biofilms,

e.g. to monitor their pH-gradients (Babauta, et al., 2011), but has not yet been exploited for the

quantification of biofilm biomass for different electrode potentials.

Page 71: PhD Thesis Alessandro Carmona2012

-50-

3.2 Materials and methods

3.2.1 General conditions

All chemicals were of analytical or biochemical grade and were purchased from Sigma-Aldrich

and Merck. If not stated otherwise, all potentials provided in this article refer to the Ag/AgCl

reference electrode (sat. KCl, 0.195 V vs. SHE). All microbial experiments were performed

under strictly sterile conditions.

3.2.2 Cell cultures and media

Sterilized LB broth (Ringeisen, et al., 2006), and minimal media (Bretschger, et al., 2007) (18

mmol/ L sodium lactate, PIPES buffer 15.1 g×L-1

; NaOH 3.0 g×L-1

; NH4Cl 1.5 g×L-1

; KCl 0.1

g×L-1

; NaH2PO4∙H2O 0.6 g×L-1

; NaCl 5.8 g×L-1

; Wolfe’s mineral solution 10 mL×L-1

(Atlas,

1993); Wolfe’s vitamin solution 10 mL×L-1

(Atlas, 1993); amino acid solution 10 mL×L-1

(Bretschger, et al., 2007)) for liquid cultures and LB/agar (Merck KGaA, Germany) plates were

used for culture maintenance.

Single colonies on LB/agar plates freshly streaked from a frozen glycerol stock culture

(Coursolle, et al., 2010) of S. putrefaciens wild-type NCTC 10695 (Nealson and Myers, 1992,

Pivnick, 1955, Venkateswaran, et al., 1999, Vogel, et al., 1997) (DSM No.: 1818, DSMZ -

German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany);

were transferred to 15 mL of LB broth and incubated aerobically at room temperature while

shaking at 100 rpm (Universal shaker SM 30 A, Edmund Bühler GmbH, Germany) for 48 h

(Carmona-Martínez, et al., 2011). Afterwards 15 mL of culture were spun down at 3000 rpm

during 10 min (Heraeus Megafuge 1.0, Germany). The pellet was resuspended in 15 mL of

minimal media and transferred to a sterile Erlenmeyer flask with 185 mL of minimal media for

72 h of cultivation using the same conditions. Finally 200 mL of media were centrifugated at

3000 rpm during 10 min, the pellet was resuspended in 15 mL of minimal media and injected in

the electrochemical cell.

Page 72: PhD Thesis Alessandro Carmona2012

-51-

3.2.3 Bioelectrochemical set-up and experiments

Polycrystalline carbon rod (CP Graphite GmbH, Germany) was used as working (2.0 cm height

x 1.0 cm diameter) and as counter (7.0 cm height x 1.0 cm diameter) electrodes for the growth

and investigation of the (anodic) electrocatalytic biofilms. Carbon electrodes were glued with

stainless steel wire (AISI 304, Fe/Cr18/Ni10, Goodfellow GmbH, Nauheim, Germany) using a

two-component resin (Epoxy resin HT 2 + Hardener HT 2, HY-POXY® Systems, SC, USA)

mixed with carbon black particles (Vulcan XC-72, Cabot Corporation GMBH, Frankfurt am

Main, Germany).

All bioelectrochemical experiments were conducted under strictly sterile conditions and under

potentiostatic control, using Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE,

Sensortechnik Meinsberg, Germany). S. putrefaciens biofilms were grown in potentiostatic half-

cell experiments at six different anode potentials (-0.1, 0, +0.1, +0.2, +0.3 and +0.4 V) at 30 °C

(Owen, et al., 1978) – see also Figure S10-1. To assure comparability and reproducibility up to

six electrodes were measured simultaneously in one electrochemical cell using an Autolab 30

potentiostat (Ecochemie, The Netherlands) equipped with six array channels. The biofilm

growth was performed in semi-batch chronoamperametric experiments at different applied

potentials with a regular Shewanella cells addition procedure (Baron, et al., 2009) and media

replacement as described below. For these potentiostatic biofilm growth experiments, initial

aerobic conditions were assured according to previous reports (Biffinger, et al., 2009).

Therefore, before adding the growth media into the electrochemical cell (Rosenbaum, et al.,

2010a, von Canstein, et al., 2008) filtered air was pumped into the fresh minimal media using a

fish pump (Elite air pump 799, Rolf C. Hagen Corp., MA, USA) per 400 mL for more than 1 h.

Fresh electron donor and nutrients were supplied about every 72 h by removing 320 mL of

culture (representing 80% of the cell volume) and replacing with fresh minimal media and fresh

Shewanella cells according to Section 3.2.2. Cyclic voltammograms were recorded for turnover

and non-turnover conditions according to (Carmona-Martínez, et al., 2011).

3.2.4 Electrochemical data processing

All data are based on experiments of 3 independent biofilm replicates and the standard

deviations are presented. The maximum current density, jmax, (calculated per projected electrode

surface area) of a batch-cycle was taken as a measure of activity and was analyzed during at

least 6 semi-batch cycles for established microbial biofilms. For in-depth data analyses of the

cyclic voltammograms the open–source software SOAS (Fourmond, et al., 2009) was used for

baseline (capacitive current) correction for non-turnover conditions.

Page 73: PhD Thesis Alessandro Carmona2012

-52-

3.2.5 Confocal Laser Scanning Microscopy

Shewanella biofilms on polycrystalline graphite electrodes were examined by CLSM after

staining with nucleic acid-specific fluorochromes. For this purpose, whole cylindrical electrodes

(~2.0 cm in exposed length) were mounted on a plastic Petri dish with silicon glue, subsequently

stained with Syto9 (Molecular Probes, OR, USA) and incubated at room temperature for 5 min

(Neu, et al., 2010). The laser microscope (SP5X, Leica Germany) was equipped with a super

continuum light source and an upright microscope. The system was controlled by the LEICA

CONFOCAL software version 2.4.1 Build 1537 (Leica, Germany). For imaging, the stained

biofilms were covered with tap water and examined with a 63x 0.9 NA objective lens. Each

Shewanella biofilm attached to the electrode was scanned at ten different locations from top to

bottom. The settings for excitation and emission/detection were as follows: excitation with the

white laser at 483 nm, detection of reflection from 475 to 495 nm and of Syto9 from 500 to 570

nm. For recording the bacterial emission signal, the lookup table "glow-over-under" was used in

order to optimally adjust signal to noise ratio and taking advantage of the full dynamic range of

the photomultiplier. Quantification was done with an extended version of ImageJ

(rsbweb.nih.gov.ij) as described elsewhere (Staudt, et al., 2004). The 8 bit data sets were

thresholded at 60. Image data sets were printed from Photoshop (Adobe).

3.3 Results and discussion

3.3.1 Bioelectrochemical current production

Figure 3-1 shows for exemplary fed-batch cycles the chronoamperometric (CA) current

production of S. putrefaciens NCTC 10695 for different applied electrode potentials. As can be

seen the maximum current density, jmax, as well as the transferred charge of each electrode

differed significantly. Since all electrodes were hosted within one vessel in order to assure

similar biological conditions for all electrodes in each experiment (see material and methods),

no coulombic efficiencies can be calculated for the individual electrode potentials.

However, when taking jmax at a given electrode potential a clear trend can be observed. As

Figure 3-2 shows the current density increases from less than 3 µA cm-2

at -0.1V up to ~12 µA

cm-2

at +0.4 V.

Page 74: PhD Thesis Alessandro Carmona2012

-53-

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.

Figure 3-2 Chronoamperometric current density of S. putrefaciens as function of the applied

electrode potential.

0 1 2 3 4 5 6 7 8

-2

0

2

4

6

8

10

12

14

16

+0.4

+0.3

+0.2

+0.1

0.0

-0.1

AAA

j ma

x/

A c

m-2

time/ days

BBB

-0.1 0.0 0.1 0.2 0.3 0.40

3

6

9

12

15

j ma

x/

A c

m-2

applied potential/ V vs. Ag/AgCl

Page 75: PhD Thesis Alessandro Carmona2012

-54-

Thus, the measured current densities are well in line with previous studies on Shewanellaceae

using plain carbon electrodes (see Table S10-1 in SI for a comparative data compilation). No

biofilms could be seen by the naked eye on all electrodes, but the solution showed significant

turbidity and a reddish pellet of bacteria was formed when spinning it down (see Fig. S1-inset).

This finding is well in accordance with previous reports on Shewanaellaceae (Babauta, et al.,

2011, Coursolle, et al., 2010, Okamoto, et al., 2011, Yang, et al., 2011), yet in contrast to pure

culture biofilms of Geobacter species or mixed culture derived biofilms dominated by

Geobacteraceae. In order to elucidate reasons for the varying electrochemical performance at

the different electrode potentials, cyclic voltammetry (CV) and confocal laser scanning

microscopy (CLSM) were employed.

3.3.2 Cyclic voltammetric analysis

Figure 3-3 shows the CVs of the electrodes under turn-over conditions, i.e. in the presence of the

electron donor lactate. All CVs show an increasing bioelectrocatalytic activity for all potentials

more positive than 0.0V. All CVs possess a half wave potential of about ~10 mV (as can be

derived from the maxima of the respective first derivatives (Fricke, et al., 2008, Marsili, et al.,

2008b)). This potential can commonly be ascribed to DET-proteins (see e.g. (Hartshorne, et al.,

2007, Shi, et al., 2006)). In this course it has to be noticed that this is the first voltammetric

study of S. putrefaciens NCTC 10695 (for S. putrefaciens from other sources see Table S10-2).

Thus, the results for turn-over conditions point out that the MET via soluble mediators plays

only a minor role, as the formal potentials, Ef, for these electron shuttling compounds (and thus

the corresponding half wave potential of the turn-over CV) are more negative. Noteworthy, the

riboflavin concentration of our minimal media nutrient broth was only about 0.1 μM (see

materials and methods). When this concentration was increased to 1 μM, the turn-over CV-

shape changed significantly and clearly illustrated that the riboflavin based MET was dominant

in these conditions (see SI Figures S10-2 and S10-3). Thus, as no significant MET associated

bioelectrocatalysis was found after more than two weeks of continuous cultivation, our results

suggest that S. putrefaciens NCTC 10695 did not synthesize relevant amounts of electron shuttle

compounds. This finding is in contrast to other species of the Shewanella family like S.

oneidensis MR-1 (Jiao, et al., 2011, Marsili, et al., 2008a, von Canstein, et al., 2008). We

suggest to ascribe this finding to the particular growth conditions especially i) the almost anoxic

Page 76: PhD Thesis Alessandro Carmona2012

-55-

behavior and ii) low mediator concentration in the medium and thus fast establishment of a

biofilm at the electrode.

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

.

In order to study the biofilm associated direct electron transfer processes in more detail, CVs for

non turn-over (lactate depleted) conditions were recorded (see Figure 3-4 A for the raw data and

Figure 3-4 B for the baseline corrected traces). It can be clearly seen that the non turn-over CVs

for the selected electrode potentials differed significantly. As all electrodes were hosted within

one vessel, these differences cannot be attributed to the suspended microbial cells, but to the

individual biofilms. Noteworthy, no CV signals were detected for electrodes poised at -0.1V and

Page 77: PhD Thesis Alessandro Carmona2012

-56-

0.0V during biofilm growth, which can clearly be attributed to the absence of the interaction

with bacteria on the electrode surface (vide infra).

As Figure 3-4 shows, the CV analysis of all further electrodes revealed that the formal potential

of the redox-active species is similar for all growth potentials (about -60 mV, see also Table

S10-3). This value is close to the maximum in the first derivatives of the turn-over CVs (Figure

3-3) showing that the detected redox couple is responsible for the bioelectrocatalytic electron

transfer. As can be seen from Figure 3-4 the peak heights and areas of the oxidation and

reduction peaks are increasing with more positive electrode potential during biofilm growth.

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.

Page 78: PhD Thesis Alessandro Carmona2012

-57-

Deriving from the data exemplary shown in Figure 3-4, Figure 3-5 depicts the peak areas and

peak heights of the respective oxidation and reduction peak as function of the applied potential

during CA. As the peak area can be considered to be a measure of the amount of a redox species

on the electrode surface (Wang, et al., 2002a, Wang and Wang, 2004), this analysis shows that

with increasingly positive potential during bacterial growth more DET-protein is deposited on

the electrode surface. As the increasing amount of DET protein can be either attributed to an

increasing cell number or to an increase in the protein amount per individual cell confocal laser

scanning microscopy (CLSM) was exploited to study these biofilms at the morphological level.

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.

As already discussed above, the detected formal potential is close to but not identical to those of

MtrC and OmcA commonly reported in literature for different experimental conditions, e.g. on

isolated proteins (Hartshorne, et al., 2007). Here it should be noticed that additional cyclic

voltammetry experiments conducted on S. putrefaciens cells suspensions (data not shown) did

not provide redox peaks in any condition tested, i.e. 1) bacteria growing microaerophilically in

LB broth; 2) cells growing aerobically in minimal media (Bretschger, et al., 2007) and 3) cells

-0.1 0.0 0.1 0.2 0.3 0.4

-1

0

1

2

3

pea

k h

eig

ht/

A

cm

-2

applied potential / V vs. Ag/AgCl

-0.2

0.0

0.2

0.4

pea

k a

rea/

C

cm

-2

Page 79: PhD Thesis Alessandro Carmona2012

-58-

growing anaerobically in (Miller and Wolin, 1974) minimal media (Bretschger, et al., 2007,

Marsili, et al., 2008a, Meitl, et al., 2009) using sodium fumarate as electron acceptor.

3.3.3 Biofilm imaging using confocal laser scanning microscopy (CLSM)

Figure 3-6 Maximum intensity projection of confocal laser scanning microscopy data sets

showing Shewanella putrefaciens biofilms grown on electrode surfaces at different applied

Page 80: PhD Thesis Alessandro Carmona2012

-59-

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.

Figure 3-7 Biofilm quantification of Shewanella putrefaciens biofilms grown on electrode

surfaces at different applied potentials.

In order to study the biofilm formation at the electrode surface exemplary CLSM measurements

were performed. As Figure 3-6 shows the number of cells (Franks, et al., 2009) seems to

increase with more positive electrode potential. This is confirmed by a biomass quantification

based on the CLSM imaging and subsequent digitital image analysis (Figure 3-7). Please note

that the high numeric value in Figure 3-7 for -0.1V can only be attributed to an inhomogeneous

biofilm growth and biofilm sloughing. This effect needs further consideration in future studies

with focus on biofilm development over time. The data is showing a linear increase of biomass

with more positive electrode potential. Nevertheless the relatively high standard deviation is

obvious and well known from other studies, e.g. (Lewandowski, et al., 2004), indicating a non-

uniform (rough) biofilm coverage. Interestingly, as Figure 3-6 shows, no complete coverage of

the electrode surface and decent biofilm formation was detected for all applied electrode

potentials. This finding is well in line with previous studies on Shewanella (Babauta, et al.,

2011, Coursolle, et al., 2010, Okamoto, et al., 2011, Yang, et al., 2011), but in clear contrast to

Geobacter species (Franks, et al., 2009, Williams, et al., 2009).

-0.1 0.0 0.1 0.2 0.3 0.4

0

1000

2000

3000

4000

applied potential/ V vs. Ag/AgCl

bio

ma

ss/

m3

Page 81: PhD Thesis Alessandro Carmona2012

-60-

Therefore, and in combination with the dominance of DET and planktonic cell growth, one may

conclude that attachment of S. putrefaciens cells was not permanent, indicating an intermittent

cell-electrode contact for electron release (Harris, et al., 2010). Furthermore, this shows clearly

the attraction of the more positive potential for biofilm formation, which is in line with a report

on the bacterial movement to electrodes, termed “electrokinesis” (Harris, et al., 2010).

3.4 Conclusions

This study on the electroactive microorganism S. putrefaciens NCTC 10695 shows that:

The chronoamperometric current generation, jmax, is a function of the applied electrode

potential; it increases from 3 µA cm-2

at -0.1V up to ~12 µA cm-2

at +0.4 V.

The amount of biomass deposited on the electrode is a function of the electrode potential

and steadily increase by factor 5 from 0V to +0.4 V.

For the range of applied electrode potentials studied the direct electron transfer (DET) is

dominating.

The bioelectrocatalytic current generation, the CV signal of the DET protein and the

biomass coverage are clearly linked by the applied electrode potential during biofilm

growth.

Interestingly, for electrode potentials more positive than the formal potential of the active site (-

60mV) the current density and biofilm formation increase. The same phenomena is found for the

turn-over CVs not showing a plateau of current density, which is in contrast to species like

Geobacter (Fricke, et al., 2008).

Most of the results found for S. putrefaciens NCTC 10695 are well in line with literature,

however some vary. These variations might be attributed to i) the specific nature of the strain S.

putrefaciens NCTC 10695 or ii) the specific growth conditions and thus may change in other

environments, e.g. presence vs. absence of O2 in the medium or constant vs. varying electrode

potentials. Therefore, they are in a row of studies on Shewanella species, providing snapshots

but not allow drawing conclusive pictures. Thereby, this study highlights exemplary the need for

a unified framework of biofilm growth and operation of electroactive biofilms, like

Shewanellaceae (Harnisch and Rabaey, 2012) in order to extract more (universal) information

out of the gained data.

Page 82: PhD Thesis Alessandro Carmona2012

-61-

CHAPTER IV CHAPTER IV

4 Spectroelectrochemical analysis of intact microbial

biofilms of Shewanella putrefaciens for sustainable energy

production

4.1 Introduction

The ability of some microbes to respire insoluble metal oxides and electrodes relays on the

peculiar extracellular electron transfer (ET) strategy they evolved. In fact this mechanism by

which microorganisms generate energy for cell growth and maintenance (Hernandez and

Newman, 2001), allows the bacteria to transfer electrons from their internal metabolism through

a chain of trans-membrane proteins to insoluble metal electron acceptors placed outside the

microbial cell (Fig. 4-1). In the early 1990s, environmental microbiologists came to realize the

importance of microbial ET of 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 multidisciplinary way not only to study the fundamentals of microbial ET but also to

apply this concept in the so-called Bioelectrochemical systems (BESs) (Rabaey, 2010), e.g., for

the production of: i) electricity (Logan, et al., 2006), ii) hydrogen as a clean fuel (Logan, et al.,

2008) and iii) useful chemicals (Rabaey and Rozendal, 2010) such as hydrogen peroxide, among

other possible applications.

Page 83: PhD Thesis Alessandro Carmona2012

-62-

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.

The scientific information available on microbial ET has rapidly increased mainly due to the

discover of two model bacteria capable of reducing insoluble metal electron acceptors:

Shewanella oneidensis MR-1 (Myers and Nealson, 1988) and Geobacter metallireducens GS-15

(Lovley and Phillips, 1988). Whereas the electrochemical analysis of the ET mechanisms for

Geobacter is well established (Fricke, et al., 2008, Marsili, et al., 2010, Millo, et al., 2011,

Srikanth, et al., 2008), Shewanella has been shown to possess more complex bioelectrochemical

behavior.

Interestingly, Geobacter and Shewanella species rely upon different microbial ET mechanisms,

i.e. direct and a combination of direct-mediated ET, respectively (Bretschger, et al., 2010b,

Gralnick and Newman, 2007, Hernandez and Newman, 2001, Marsili and Zhang, 2010,

Schröder, 2007, Watanabe, et al., 2009). In direct electron transfer (DET), direct electrical

contact between the bacterial cell and the electrode is provided by outer membrane cytochromes

(OMCs). These multiheme redox proteins embedded within the outer membrane of the bacterial

cell shuttle electrons between the respiratory chain and the insoluble electron acceptor through a

densely packed chain of heme groups. Recently, the DET has been proposed to occur also

through cellular appendages facing the extracellular environment (i.e., microbial nanowires).

Although these appendages have been initially observed for Shewanellaceae (El-Naggar, et al.,

Page 84: PhD Thesis Alessandro Carmona2012

-63-

2010, Gorby, et al., 2006), they have been recently found also for Geobacteraceae (Malvankar,

et al., 2011, Reguera, et al., 2005) families, where they have been proposed to sustain long-

range metallic-like ET (Malvankar et al., 2011). In mediated electron transfer (MET), the

bacteria release soluble redox mediators such as flavin or melanin, that go through a series of

reduction and oxidation processes taking place between the bacterial cell and an extracellular

insoluble compound (Turick, et al., 2002, von Canstein, et al., 2008).

It is worth noting that one mechanism does not exclude the other, in such a way that DET and

MET can function simultaneously within the same microbial community. This is the case of

Shewanella oneidensis MR-1. As recently reported by Shi and co-workers (Shi, et al., 2009) the

DET performed by S. oneidensis MR-1 depends on inner membrane cytochromes (IMCs) and

OMCs 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 periplasmic decahaem c-Cyt MtrA, the outer membrane protein

MtrB and the OMC decahaem c-Cyts MtrC and OmcA (Shi, et al., 2009). All these proteins

together form a pathway to transfer electrons from the quinone/quinol pool in the inner

membrane to the periplasm and then to the outer membrane where MtrC and OmcA can either

transfer electrons directly to the surface of electrode materials or to soluble redox mediators.

Although several studies have investigated the behavior of isolated OMCs attached on

electrodes (Eggleston, et al., 2008, Firer-Sherwood, et al., 2008b, Hartshorne, et al., 2007,

Hartshorne, et al., 2009, Meitl, et al., 2009, Pankhurst, et al., 2006, Turner, et al., 1999), or

embedded in microbial biofilms of Shewanella (Carmona-Martínez, et al., 2011, Coursolle, et

al., 2010, Liu, et al., 2011, Meitl, et al., 2009, Nakamura, et al., 2009a, Okamoto, et al., 2011,

Okamoto, et al., 2009), the role of these cytochromes in the heterogeneous ET across the

biofilm/electrode interface is far from clearly understood.

Surface-enhanced resonance Raman scattering (SERRS) is a sensitive and selective technique

able to probe surface-confined heme species in the sub-monolayer coverage (Khoa Ly, et al.,

2011). This approach, often used to study isolated proteins immobilized on nanostructured (i.e.

surface-enhanced Raman active) electrodes, is also applicable to multiheme proteins embedded

within intact electrochemical-active microbial biofilms (Millo, et al., 2011). In this case, SERRS

probes selectively the heme groups of the OMCs in the close vicinity of the electrode surface,

revealing important structural information, such as the oxidation-, the coordination-, the spin-

Page 85: PhD Thesis Alessandro Carmona2012

-64-

state, and the nature of the axial ligands of the central iron atom of these proteins. When

performed in combination with electrochemical techniques, stationary and time-resolved

SERRS reveal unprecedented insights into the thermodynamics and the kinetics of the

heterogeneous electron transfer across the bacteria/electrode interface.

The aim of this study is to achieve a better understanding of the ET in microbial biofilms of S.

putrefaciens NCTC 10695. By measuring the electrochemical and spectroscopic properties of

microbial cells embedded in their natural biofilm habitat, a more realistic picture on the natural

electron transfer will be provided.

4.2 Materials and methods

4.2.1 Materials and methods

All chemicals were of analytical or biochemical grade and were purchased from Sigma-Aldrich

and Merck. If not stated otherwise, all potentials provided in this article refer to the Ag/AgCl

reference electrode (sat. KCl, 0.195 V vs. SHE). All microbial experiments were performed

under strictly sterile conditions.

4.2.2 Cell cultures and media

Sterilized LB broth (Ringeisen, et al., 2006), and minimal media (Bretschger, et al., 2007) (18

mmol/ L Sodium lactate, PIPES buffer 15.1 g×L-1

; NaOH 3.0 g×L-1

; NH4Cl 1.5 g×L-1

; KCl 0.1

g×L-1

; NaH2PO4∙H2O 0.6 g×L-1

; NaCl 5.8 g×L-1

; Wolfe’s mineral solution 10 mL×L-1

(Atlas,

1993); Wolfe’s vitamin solution 10 mL×L-1

(Atlas, 1993); Amino acid solution 10 mL×L-1

(Bretschger, et al., 2007)) for liquid cultures and LB/agar (Merck KGaA, Germany) plates were

used for culture maintenance.

Single colonies on LB/agar plates freshly streaked from a frozen glycerol stock culture

(Coursolle, et al., 2010) of S. putrefaciens wild-type NCTC 10695 (Nealson and Myers, 1992,

Pivnick, 1955, Venkateswaran, et al., 1999, Vogel, et al., 1997) (DSM No.: 1818, DSMZ -

German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany);

were transferred to 15 mL of LB broth and incubated aerobically at room temperature while

shaking at 100 rpm (Universal shaker SM 30 A, Edmund Bühler GmbH, Germany) for 48 h

(Carmona-Martínez, et al., 2011). Afterwards 15 mL of culture were spun down at 3000 rpm

during 10 min (Heraeus Megafuge 1.0, Germany). The pellet was resuspended in 15 mL of

minimal media and transferred to a sterile Erlenmeyer flask with 185 mL of minimal media for

72 h of cultivation using the same conditions. Finally 200 mL of media were centrifugated at

Page 86: PhD Thesis Alessandro Carmona2012

-65-

3000 rpm during 10 min, the pellet was resuspended in 15 mL of minimal media and injected in

the electrochemical cell.

4.2.3 Electrochemical set-up for the growth of anodic electrocatalytic biofilms

Sealed vessels (200 mL) served as the electrochemical cell hosting the three-electrode

arrangement (Fig. 4-2). The bioelectrochemical experiments were conducted under strictly

sterile conditions and under potentiostatic control (Potentiostat/Galvanostat VMP3, BioLogic

Science Instruments, France) utilizing a three-electrode arrangement, with a silver ring working

electrode (0.7 cm2) electrochemically roughened according to the procedure described elsewhere

(Wackerbarth, et al., 1999), a polycrystalline carbon rod counter electrode (CP Graphite GmbH,

Germany, 7.0 cm height x 1.0 cm diameter) and Ag/AgCl reference electrode (sat. KCl, 0.195 V

vs. SHE, Sensortechnik Meinsberg, Germany). The carbon electrode was glued with stainless

steel wire (AISI 304, Fe/Cr18/Ni10, Goodfellow GmbH, Nauheim, Germany) using a two-

component resin (Epoxy resin HT 2 + Hardener HT 2, HY-POXY® Systems, SC, USA) mixed

with carbon black particles (Vulcan XC-72, Cabot Corporation GMBH, Frankfurt am Main,

Germany).

Figure 4-2 Electrochemical half cell set-up under potentiostatic control. Insert shows a

photograph of the nanostructured silver ring working electrode.

Page 87: PhD Thesis Alessandro Carmona2012

-66-

4.2.4 Growth of anodic electrocatalytic biofilms

The biofilm growth was performed in semi-batch chronoamperametric experiments at +0.05 V

at 30 °C (Owen, et al., 1978) with a regular Shewanella cells addition procedure (Baron, et al.,

2009) and media replacement as described below. For these potentiostatic biofilm growth

experiments, initial aerobic conditions were assured according to previous reports (Biffinger, et

al., 2009). Therefore, before adding the growth media into the electrochemical cell (Rosenbaum,

et al., 2010a, von Canstein, et al., 2008) filtered air was pumped into the fresh minimal media

using a fish pump (Elite air pump 799, Rolf C. Hagen Corp., MA, USA) per 200 mL for more

than 1 h. Fresh electron donor and nutrients were supplied about every 72 h by removing 160

mL of culture (representing 80% of the cell volume) and replacing with fresh minimal media

and fresh Shewanella cells according to Section 4.2.2.

4.2.5 Cyclic voltammetry

Cyclic voltammograms were recorded for non-turnover conditions according to (Carmona-

Martínez, et al., 2011) . Potentials were applied from -500 to +50 mV (vs. Ag/AgCl) at a scan

rate of 1 mV s-1

with continuous monitoring of the current response.

4.2.6 Electrochemical data processing

All data are based on experiments of 3 independent biofilm replicates and the standard

deviations are presented. The maximum current density, jmax, (calculated per projected electrode

surface area) was taken as a measure of activity and was analyzed during at least 2 semi-batch

cycles for established microbial biofilms. For in-depth data analyses of the cyclic

voltammograms the open–source software SOAS (Fourmond, et al., 2009) was used for baseline

(capacitive current) correction for non-turnover conditions.

4.2.7 Spectroelectrochemical set-up for SERRS measurements

Electrochemical experiments were carried out in a homemade spectroelectrochemical cell

working in the three electrode configuration and controlled by a μAutolab potentiostat (Eco

Chemie, Utrecht, The Netherlands). The three electrodes were a biofilm-coated (vide supra) Ag

ring electrochemically roughened (see 4.2.3), a Pt coil, and a Ag/AgCl (3.0 M KCl) (Dri-Ref,

WPI Berlin, Germany), acting as working, counter, and reference electrode, respectively.

4.2.8 SERRS measurements

SERRS spectra were obtained using a confocal Raman spectrometer (LabRam HR-800, Jobin

Yvon) with a spectral resolution of 2 cm-1

and an increment per data point of 0.57 cm-1

. SERR

spectra were obtained with 413 nm excitation (Coherent Innova 300 c Krypton cw-laser) with a

Page 88: PhD Thesis Alessandro Carmona2012

-67-

laser power of 1.0 mW on the sample. The laser beam was focussed by a Nikon 20x objective

with a working distance of 20.5 mm and a numeric aperture of 0.35. Further details of the

experimental set-up are given elsewhere (Millo, et al., 2011).

4.3 Results and discussion

4.3.1 Bioelectrochemical current production

The biofilms were grown at a constant potential on roughened (i.e. SER-active) silver electrodes

using 18 mM sodium lactate as substrate (see above for experimental details). These biofilms

produced a maximum chronoamperometric current density of 3.03 ± 0. 13 μA cm-2

(Fig. 4-3),

which is in good agreement with previous studies using aerobe graphite anodes (see chapter 2

and 3).

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.

The voltammetric behavior of the biofilms was monitored under non-turnover conditions (i.e.,

without sodium lactate). Fig. 4-4 shows the CV behavior of such a biofilm for non-turnover

conditions. The redox couple that is proposed to be involved in the DET (i.e. the one ascribed to

Page 89: PhD Thesis Alessandro Carmona2012

-68-

the oxidation/reduction of the heme groups of the surface-confined OMC), Ef,1 is centered at a

formal potential of -233 mV (vs. the Ag/AgCl reference electrode). The main overall shape and

peak positions of the cyclic voltammogram shown in Fig. 4-4 are very similar to those obtained

on graphite electrodes in previous studies (see chapter 3), showing that biofilm formation is not

affected by the nature of the electrode material. Furthermore, this finding reveals that the

nanostructured silver electrode and specifically the inevitable traces of AgI cations do not

provide a toxic environment for Shewanella putrefaciens, confirming recent observations by

Preciado-Flores and co-workers (Preciado-Flores, et al., 2011) who were able to grow

Shewanella oneidensis cells in the presence of silver nanoparticles and nanowires.

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.

Figure 4-5 shows the SERR spectra of a microbial biofilm grown on a roughened Ag electrode

and measured at different applied potentials in the absence of metabolic substrate. Spectral

analysis allows ascribing the SERRS signal to one or more heme species in the vicinity of the

electrode surface. Admittedly, the fingerprint region differs from that of a c-type heme, lacking

the typical band centered at 418 nm ascribed to the CCC vibrational mode of the heme (not

shown). Therefore, the type of heme cannot be unambiguously assigned. Moreover, the

frequency of the oxidation marker band 4 (in Fig. 4-5 at 1374 nm) has been found to vary

significantly. Although the high frequency values clearly show that the heme is oxidized, the

origin of the frequencies scattering obtained for different samples (1370-1380 cm-1

) and for the

same sample measured on two different days (1375-1380 cm-1

) is still unknown. Remarkably,

Page 90: PhD Thesis Alessandro Carmona2012

-69-

no correlation with the sample treatment and/or preparation has been observed. Interestingly,

this frequencies scattering has not been observed for the other vibrational modes (i.e the 3, 2,

and 10). These frequencies are in agreement with a six-coordinated low-spin Fe atom with two

His residues acting as axial ligands (Oellerich, et al., 2002).

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).

As shown in Figure 4-5, spectra recorded at two different poised potentials are very similar.

Remarkably, the spectrum at -475 mV does not display the expected band downshift of the 4

upon electrochemical reduction. This finding is indicative that the heme species detected by

SERRS – probably the heme groups of surface-confined OMCs – do not promote the electron

transfer. OMC reduction has been attempted by applying a negative potential in the presence of

(i) O2, (ii) the soluble redox mediator riboflavin, (iii) O2 and riboflavin together. In all cases no

reduction has been observed. Full OMC reduction has been achieved by exposing the biofilm at

open circuit potential to the soluble reducing agent dithionite (Na2S2O4). This procedure has

lead to the immediate OMC reduction, as proven by the downshift of the 4 to 1360 cm-1

(not

Page 91: PhD Thesis Alessandro Carmona2012

-70-

shown). However, after this harsh chemical treatment, the biofilm lost its capability of

generating electrical current. In fact, chronoamperometric experiments performed in the

presence of lactate did not show the recovery of catalytic activity.

4.4 Conclusions

The experiments here presented, performed on microbial biofilms of S. putrefaciens NCTC

10695, have shown that OMCs do not contribute to the heterogeneous ET across

bacteria/electrode interface. These studies have been performed on biofilms grown on

nanostructured Ag electrodes at the poised potential of +50 mV (vs. Ag/AgCl). Although these

conditions allow the formation of a biofilm on the Ag electrode, they may have a negative

impact on the amount of OMCs expressed by the bacteria (see chapter 3). In fact, optimal

biofilm growth requires more positive potentials. However, these conditions cannot be met by

the Ag substrate, which undergoes oxidation at potentials higher than +150 mV (vs. Ag/AgCl).

The development of novel analytical strategies to overcome this limitation is presently under

evaluation in our groups. Furthermore, under the supervision of Dr. Millo, I will perform further

experiments at the Vrije Universiteit Amsterdam that will allow us achieve a better

understanding of the electron transfer in microbial biofilms of S. putrefaciens.

Page 92: PhD Thesis Alessandro Carmona2012

-71-

CHAPTER V CHAPTER V

5 Electrospun and solution blown three-dimensional

carbon fiber nonwovens for application as electrodes in

microbial fuel cells

5.1 Introduction

There is an ever increasing interest in the research community and industries in electrospinning

and solution blowing of microfiber, nanofiber and nonwovens for applications such as

biomedical (Agarwal, et al., 2008, Reneker, et al., 2007), tissue engineering (Pham, et al., 2006,

Wise, et al., 2009), drug release (Gandhi, et al., 2009, Srikar, et al., 2008, Wang, et al., 2010),

agriculture (Hellmann, et al., 2011), etc. Recently high-temperature electrospun nonwovens

were used as separators in lithium-ion batteries (Bansal, et al., 2008). Typical electrospun

nonwovens represent themselves thin, practically two-dimensional, sheets of low permeability.

For a number of applications, e.g. tissue engineering, three-dimensional, fluffy, nonwovens

could be of great interest.

Preparation of three-dimensional nanofiber nonwovens by remodeling of nanofiber yarn

electrospun onto water surface was recently reported in biomedical context (Teo, et al., 2008).

Three-dimensional multi-layered cell–nanofiber constructs for tissue engineering were also

prepared by a kind of layer-by-layer electrospinning (Yang, et al., 2009). The new capabilities

of the preparation techniques and versatility of the resulting nonwoven materials raise the

question if bioelectrochemical systems (BES) like microbial fuel cells can benefit from these

developments and their performance can be increased further more. The great majority of

current microbial BES utilizes electroactive microbial biofilms as electrocatalyst to facilitate the

anodic substrate oxidation.

Key players in such biofilms are electroactive bacteria like Geobacter or Shewanella species

that are often also referred to as electricigens or exoelectrogens (Gorby, et al., 2006, Logan,

2009, Lovley, 2006, Rabaey, et al., 2010, Rabaey, et al., 2007). Exploiting electroactive bacteria

in BES, e.g. in microbial fuel cells or electrolysis cells (Rabaey, et al., 2010), promises a great

potential in the context of sustainable energy supply and handling. A major challenge in this

context is an increase in the performance of such systems. During the past decade the average

Page 93: PhD Thesis Alessandro Carmona2012

-72-

current densities of biofilm anodes have already increased significantly from milliampere per

square metre level to between 7 and 10 A m-2

. This increase can be mainly attributed to the

improvement of the microbial biofilms, e.g. via sophisticated enrichment and acclimatization

procedures (Kim, et al., 2004, Liu, et al., 2008, Rabaey, et al., 2004). Recent experimental

results and theoretical considerations suggest that a further improvement above 10 A m-2

may be

hampered by limitations imposed by kinetics associated with electron and proton transfer as well

as substrate diffusion within the biofilm (Torres, et al., 2010, Torres, et al., 2008). Since the

improvement of the biological component becomes increasingly difficult, the improvement or

even the tailoring of the electrode materials becomes an important task. In the past, such

electrode tailoring has already been shown to be successful for microbial fuel cell based on

suspended bacterial cultures (Rosenbaum, et al., 2006, Schröder, et al., 2003). One strategy to

enhance the performance of biofilm anodes is the improvement of the electrode surface

properties by surface treatment procedures such as ammonia treatment, polymer modification or

surface oxidation (Cheng and Logan, 2007, Scott, et al., 2007, Wang, et al., 2009). These

measures are likely to improve biofilm–electrode interactions and thus the rate of electron

transfer. A further, promising path is the increase of the active surface area, for example by

means of brush (Logan, et al., 2007) or fiber electrodes e.g. ref. (Liu, et al., 2010c), and 3D

electrode materials e.g. ref. (Zhao, et al., 2010b). This path already delivers promising results as,

for example in ref. (Zhao, et al., 2010b), which combines a 3D electrode structure and a

conductive polymer as an immobilized mediator. The experiments of ref. (Zhao, et al., 2010b)

yielded the highest reported current density of a biofilm electrode (about 24 A m-2

). The present

work aims to exploit high surface area electrospun and solution-blown carbonized nonwovens to

further enhance the current density of BESs anodes. Three different fiber mat materials

(nonwovens) were studied in comparison to conventional polycrystalline graphite and carbon

felt, i.e.: i) 3D porous carbon fibers, produced by gas-assisted electrospinning (hereafter denoted

as GES-CFM); ii) Electrospun carbon fibers (ES-CFM); and iii) Solution-blown carbon fibers

(SB-CFM). The latter two materials were additionally modified to incorporate 15% carbon black

(CB) to increase porosity and conductivity of the resulting nonwovens. These modified

materials are denoted as ES-CFM15%CB and SB-CFM15%CB. In order to study and compare

the bioelectrocatalytic substrate oxidation at all above-mentioned electrode materials,

preselected wastewater-derived mixed culture biofilms were grown under potentiostatic control.

The bioelectrocatalytic performance was then analyzed in semi-batch, chronoamperometric

experiments by measuring the projected (geometric) current density at the respective materials.

Page 94: PhD Thesis Alessandro Carmona2012

-73-

5.2 Materials and methods

5.2.1 Carbon fiber preparation

Three techniques for the preparation of three-dimensional carbon fiber mats (CFM) were

employed for this study: electrospinning (see Fig. 5-1A, samples labeled ES), solution blowing

(SB) and gas-assisted electrospinning (GES).

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.

5.2.1.1 Gas-assisted electrospinning carbon fiber mat (GES-CFM)

A solution of poly(acrylonitrile-co-itaconic acid-co-butyl acrylate) (monomer ratio AN/IA/BA =

46/3/1; [η]25°C = 2.4 dL g-1

measured in DMAc) 11 weight% in DMSO/DMF/acetone (16/15/5)

was used for air-assisted electrospinning. The electrode distance was 100 cm between the

electrodes at a voltage of 40 kV m-1

. The carbonization process was performed in a high-

temperature furnace using the following protocol:

Page 95: PhD Thesis Alessandro Carmona2012

-74-

i) Heating up to 230°C at a rate of 20°C min-1

in air and annealing for 2 h for stabilization of the

precursor ultrafine fibers; ii) Heating up to 350°C at a rate of 5–10°C min-1

in nitrogen

atmosphere and annealing at 350°C for 20 min; iii) Heating up to 750°C at a rate of 3–5°C min-1

in nitrogen atmosphere and annealing at 750°C for 20 min; and iv) Heating up to 1000°C at a

rate of 3–5°C min-1

in nitrogen atmosphere and annealing at 1000°C for 1 h to complete the

carbonization process.

5.2.1.2 Electrospun carbon fiber mat (ES-CFM)

12% polyacrylnitrile (PAN) solutions containing 5, 7, 9, 11 and 15 wt% of carbon black (CB)

nanoparticles were electrospun. The resulting fiber mats had a very loose three-dimensional

structure with high porosity (with pores approximately from ten to hundreds of micrometres in

comparison with the ordinary electrospun fiber mats with pores of about 3 to 5 mm). The

resulting PAN fiber mats were placed in a furnace and stabilized in air for 20 min at 270°C, then

carbonized in nitrogen at 1100°C (the ramp rate was 4°C min-1

between the room temperature,

280°C, and 1100°C plateaus).

5.2.1.3 Solution-blown carbon fiber mat (SB-CFM)

Blends of PAN solutions with CB were solution blown as described in ref. (Sinha-Ray, et al.,

2010). The blends were prepared using the following steps. Initially a 12% PAN solution in

DMF was prepared. Then, 1 g of 12 wt% PAN solution, 0.12 g of CB and 4 g of DMF in a 20

mL vial were sonicated for 45 min. During sonication CB clusters were broken, dispersed and

coated by PAN, which prevented coalescence of particulates. Then, 4.32 g of DMF and 0.68 g

of PAN were added to the vial and kept on a hotplate overnight under permanent stirring.

In this way a blend of 8 wt% PAN solution with 15 wt% CB was prepared and solution blown

(Sinha-Ray, et al., 2010). The carbonization was achieved by heating of the PAN fibers in open

air at 350°C for 3 h and then in nitrogen atmosphere at 1050°C for 1 h.

Porosity data are provided as a fraction of the void volume in the total volume:

total

solid

total

void

V

V

V

V 1 Equation 5-1

Here, Vvoid is the volume of pores, Vsolid is volume of solid, Vtotal is the total or bulk volume of

material including the solid and pore volume. The volume of the fiber material can be expressed

as V = Aδ, with A being the geometric surface area and δ the equivalent thickness of the fully

compressed fiber mat.

Page 96: PhD Thesis Alessandro Carmona2012

-75-

Using a constant geometric surface area, Equation 5-1 can be written as Θ = 1 - δsolid/δtotal. Here,

δtotal represents the effective thickness of the porous fiber mat, δsolid is the equivalent thickness of

the fully compressed solid material calculated using the mass, m, of the fiber mat and the density

of graphite, ρ, as δsolid = m/ρA.

5.2.2 Electrode preparation

All above-mentioned materials were utilized as electrodes for the growth and investigation of

(anodic) electrocatalytic biofilms. For electrode preparation, either the electrode materials

prepared in this work or commercial carbon felt (Weichfilz, SIGRATHERM, SGL Carbon

GmbH, Meiningen, Germany). They were cut into 1x2 cm2 pieces and glued onto graphite foil

paddles (serving as an inert electrode backbone material; Chempur©, Karlsruhe, Germany)

using a two-component resin mixed with carbon black particles (Vulcan XC-72). Polycrystalline

carbon was used as carbon rods (CP Graphite GmbH, Germany).

5.2.3 Bioelectrochemical experiments

All bioelectrochemical experiments were conducted under strictly anoxic conditions and under

potentiostatic control. The potentials provided in this manuscript refer to the Ag/AgCl reference

electrode (sat. KCl, 0.195 V vs. SHE, Sensortechnik Meinsberg, Germany). All microbial

biofilms were grown in potentiostatic half-cell experiments at 0.2 V at 35°C (Patil, et al., 2010).

To assure comparability and reproducibility up to six electrodes were measured simultaneously

in one electrochemical cell (cf. Fig. 5-1B).

For this purpose, an Autolab 30 potentiostat (Ecochemie, the Netherlands) equipped with six

array channels was employed. The bacterial growth medium was prepared as reported in ref.

(Liu, et al., 2008) with acetate serving as a substrate of the medium (pH 6.8). The bacterial

source for the primary biofilm formation was wastewater from the wastewater treatment plant

Steinhof, Braunschweig.

The reported current densities refer to secondary, i.e. preselected, biofilms (gained by a

procedure as described in ref. (Liu, et al., 2008). They are based on three independent biofilm

replicates with at least two feeding cycles per biofilm, exhibiting a reproducible maximum

current density. Cyclic voltammograms were recorded for turnover and non-turnover conditions

according to ref. (Fricke, et al., 2008).

Page 97: PhD Thesis Alessandro Carmona2012

-76-

5.3 Results and discussion

5.3.1 Biocatalytic current generation at modified carbon electrodes

Table 5-1 summarizes the projected (geometric) current density data and physical parameters of

the tested electrode materials. The current density data correspond to the maxima of the

respective semi-batch experiment (Fig. 5-2), averaged over at least three independent

experimental sets. Table 5-1 illustrates that conventional polycrystalline carbon graphite shows

the lowest bioelectrocatalytic current density of all tested samples. The corresponding current

value of 13 A m-2

is still quite high. This is explained by the fact that in the present work we use

preselected (secondary) biofilms with an optimized performance (Liu, et al., 2008), in

combination with the reaction temperature of 35°C (Patil, et al., 2010).

Table 5-1 Cumulative data on electrocatalytic current densities obtained at different electrode

materials. The substrate was 10 mM Sodium acetate.

Material Current densitya /

A m-2

Specific weight /

g m-2

Specific current

density/ mA g-1

Polycrystalline graphite 13 NA NA

Carbon Felt 16 333 48

GES-CFM 30 42 714

ES-CFM 21 126 243

ES-CFM15%CB 15 88 172

SB-CFM 17 437 56

SB-CFM15%CB 21 183 128

aProjected (geometric) current density, NA—not applicable.

Carbon felt revealed a higher (by 23%) performance than polycrystalline graphite. The highest

current density in this study was obtained with GES-CFM. As illustrated in Fig. 5-2, biofilms

grown on GESCFM-electrode delivered maximum current densities of 30 A m-2

. To the best of

our knowledge these are the highest current densities so far achieved with an electroactive

microbial biofilm.

The other material types, ES-CFM and SB-CFM, also yielded current densities higher than

commercial carbon felt, and up to 21 A m-2

(Table 5-1). Interestingly, the addition of carbon

black so far did not deliver unambiguous results. In the case of the solution blown material, SB-

CFM, addition of 15% CB increased the current density from 17 A m-2

to 21 A m-2

.

Page 98: PhD Thesis Alessandro Carmona2012

-77-

On the other hand, the electrospun ES-CFM did not benefit from adding carbon black. In the

latter case the current density decreased from 21 to 15 A m-2

. The reason for this behavior is not

known so far and will require further investigation.

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.

The excellent performance of GES-CFM, the porous carbon mat produced by gas-assisted

electrospinning, is realized at extremely low specific weight (see Table 5-1). In comparison to

commercial carbon felt (specific weight 333 g m-2

) that of GESCFM was decreased by 87%

(specific weight 42 g m-2

). GES-CFM further possesses an extremely low effective density of

about 18 kg m-3

-which is an order of magnitude lower than that of conventional carbon-based

materials, such as carbon felt (about 100–180 kg m-3

).

5.3.2 Analysis of electroactive biofilms grown at modified carbon electrodes with

Scanning electron microscopy

The high porosity of the material is achieved by enhanced electric fiber–fiber repulsion and

mechanical restrictions when layers of fibers are deposited one by one. Further, the presence of

Page 99: PhD Thesis Alessandro Carmona2012

-78-

a gas flow disturbs the stable electrospinning jet and causes the fiber to form loose fiber

aggregates. Due to the high solvent content during the preparation these fiber aggregates form

interconnections (Fig. 5-3E), which lead to a high mechanical stability as well as a high

electronic conductivity of the fiber mats.

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.

Page 100: PhD Thesis Alessandro Carmona2012

-79-

Thus, the excellent bioelectrocatalytic performance of this material can be attributed to a

structure that provides a habitat for the growth of electroactive bacteria up to a maximum

density supplemented by efficient substrate supply through the open pore structure. The

interconnections between the individual fibers of the nonwoven allow the formation of cross-

linked three-dimensional biofilms (Fig. 5-3D) that benefit from an optimum electron transfer

and conduction.

The origin of the performance differences between the different electrospun and solution blown

materials is so far not fully understood. All materials possess an extremely high porosity of up

to 99%. Porosity represents the fraction of void space in the material and is of utmost

importance. Indeed, high porosity not only lowers the amount of material to a minimum but also

maximizes penetration of microorganisms and the diffusional substrate supply. However,

parameters like the occurrence of cross-linking points (cf. Fig. 5-3E) may represent a key factor

that lead to an additional performance gain.

5.3.3 Cyclic voltammetry of electroactive biofilms grown at modified carbon electrodes

Cyclic voltammograms were recorded under turnover as well as non-turnover conditions in

order to study a potential effect of the electrode material on the mechanism and thermodynamics

of the biocatalytic substrate conversion. Fig. 5-4 depicts cyclic voltammograms of the

electroactive biofilms at GES-CFM under turnover as well as non-turnover conditions. The

results are typical for wastewater-derived electroactive biofilms and biofilms of Geobacter

sulfurreducens.

Thus, the cyclic voltammograms of the biofilms under non-turnover conditions reveal redox

systems with an average formal potential of -329 mV, which is in accordance with the literature

data. In particular, in previous studies, formal potentials of -324 mV were achieved for

secondary mixed culture biofilms (Liu, et al., 2008) and -331 mV for biofilms of G.

sulfurreducens (Fricke, et al., 2008), both grown at polycrystalline graphite.

Page 101: PhD Thesis Alessandro Carmona2012

-80-

In light of the recent experimental proof that G. sulfurreducens represent a strongly dominating

species in acetate-grown wastewater-derived biofilms (Harnisch, et al., 2011), the present

voltammetric results confirm that the thermodynamics of bioelectrocatalytic energy conversion

is not determined or affected by the fiber electrode material but is fully governed by the

extracellular microbial electron transfer (e.g., by the redox potential of the outer membrane

cytochromes).

This is an important issue since any deterioration of the thermodynamics by the introduction of

additional redox cascades, e.g., via the use of immobilized redox mediators, would lower the

electrochemical energy gain and thus the energy efficiency of a potential bioelectrochemical

device.

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

.

Page 102: PhD Thesis Alessandro Carmona2012

-81-

5.4 Conclusions

In conclusion, it has been shown that three-dimensional electrospun and solution-blown carbon

fiber nonwovens represent highly interesting and promising electrode materials for microbial

fuel cell applications. They combine the use of a minimum amount of carbon and great

performance. Thus, current densities of up to 30 A m-2

were achieved, which represent the

highest values demonstrated at electroactive biofilms to date. These current densities were

achieved without chemical surface modification, which may represent an advantage with respect

to longevity.

Based on this initial study, further and systematic investigations are required to fully exploit the

potential of this class of materials. A special emphasis of further investigations must be on tests

designed to elucidate the long-term behavior of the new electrode materials and the resistivity

against clogging of the pore structures. Further, a theoretical model is under investigation to

correlate and predict structure–property relationships of this new class of BES anode materials.

Page 103: PhD Thesis Alessandro Carmona2012

-82-

CHAPTER VI CHAPTER VI

6 Electrospun carbon fiber mat with layered architecture

for anode in microbial fuel cells

6.1 Introduction

Bioelectrochemical systems like Microbial fuel cells (MFCs), considered to be a green energy

conversion technology, have recently attracted appreciable attention for the conversion of

chemical energy to electricity (Logan, 2009, Logan and Regan, 2006b, Rabaey and Verstraete,

2005). The creative idea of MFCs originated from the discovery that microbial biofilms can be

adapted to extracellular electron transfer (Rabaey, et al., 2010). Numerous studies have found

that the key players in these biofilms are electroactive bacteria (Logan, 2009, Logan and Regan,

2006a), like members of the Geobacter (Harnisch, et al., 2011, Lovley, 2006) or Shewanella

(Gorby, et al., 2006) families.

The performance of MFCs, to a large extent, depends on the density of electrochemically active

biofilms on the anode. Most of the literature reported the use of two-dimensional MFC anodes

(Adachi, et al., 2008, Qiao, et al., 2008, Qiao, et al., 2007). The 2D nature of anode allowed

biofilms to grow only in one direction and made a layer on the surface. Some new materials,

such as carbon nanotube/polyaniline composite (Qiao, et al., 2007), Titanium

dioxide/polyaniline composite (Qiao, et al., 2007), and mediator mobilized carbon (Adachi, et

al., 2008) have been used for anode in MFCs and generated exciting current density.

Thus, e.g. in ref. (Adachi, et al., 2008) a current density up to 1.2 mA cm-2

has been generated

by using 9,10-anthraquinone-2,6-disulfate modified graphite felt. Carbon materials with three-

dimensional porous architecture have also been used for anodes in MFCs to increase active

surface for biofilm growth, like reticulated vitreous carbon (He, et al., 2005), graphite fiber felt

(Chaudhuri and Lovley, 2003, Liu, et al., 2010c, Zhao, et al., 2010b) and brush materials

(Logan, et al., 2007) and very promising results have been obtained. Especially for large scale

applications, e.g. wastewater treatment and the accompanying MFC up-scaling, the increase of

the overall MFC performance is still a big task.

Electrospinning is a unique process that effectively produces small fibers with diameter ranging

from a few nanometers to several microns (Greiner and Wendorff, 2007). Electrospun fiber mats

Page 104: PhD Thesis Alessandro Carmona2012

-83-

with advantages of small diameter and high specific surface area have been widely used in many

fields, which were highlighted in many review articles (Greiner and Wendorff, 2007, Thavasi, et

al., 2008). Carbon nanofiber mats (CFMs) obtained by carbonization of electrospun

polyacrylonitrile (PAN), showed a high porosity, e.g. >85%. Because of their high surface area

and porosity, the carbonized electrospun fiber mats (Hou and Reneker, 2004, Wang, et al.,

2002b, Zhou, et al., 2009, Zussman, et al., 2005) have been used as electrodes in

electrochemical cells (Guo, et al., 2009, Ji and Zhang, 2009, Kim, et al., 2007, Kim and Yang,

2003, Kim, et al., 2006). Electrospun CFMs (ECFMs) could also be used as high performance

anode in microbial fuel cells. In Chapter 5, three-dimensional ECFM prepared by gas-assisted

electrospinning, has been proved to be an efficient anode in MFCs and generated the highest

anodic current density of 3.0 mA cm-2

known - to the best of my knowledge - till date (Chen, et

al., 2011). This high anodic current density has been believed to be attributed to the large

surface area and ultrahigh porosity for growth of high density biofilm.

In this Chapter, the concept of continuous, layered anode biofilms for microbial

bioelectrochemical systems is introduced. Three-dimensional CFMs with layered architecture

(hereafter denoted as layered-CFMs) were prepared to grow continuous, layered electroactive

biofilms with high density. Natural cellulose paper (NCP) was used as support for layer-by-layer

electrospinning (LBL-electrospinning) of PAN fiber layers. The layered-CFM has an

architecture of alternating cellulose-based and electrospun PAN-based carbon fiber layers, and

shows an ultrahigh porosity of 98.5%. The layered-CFM was used as MFC anode and the

biofilm growth characteristics were investigated.

6.2 Materials and methods

6.2.1 Carbon fiber preparation

10 wt% PAN (Mw=210 k) solution in dimethylformamide (DMF) was prepared for

electrospinning. The typical conditions used for electrospinning were electric field of the order

of 60 kV m−1

, voltage of 10 kV, and the distance between two electrodes was 20 cm. The

feeding rate of solution was 0.1 mL min−1

. Layered NCP-PAN fiber mat of ten layers (numbers

of PAN fiber layers) was prepared by layer-by-layer electrospinning of PAN fibers onto thin

NCP (in the form of tissue paper). Each PAN fiber layer was electrospun for about 5 min. All

fiber mat samples were dried in vacuum at 60 °C for subsequent carbonization.

The electrospun fiber mats and NCP were sandwiched between two graphite plates and

carbonized in a tubular stainless steel reactor by using the following protocol: 1) in air

Page 105: PhD Thesis Alessandro Carmona2012

-84-

atmosphere, heating up to 230°C at a rate of 2°C min−1

and annealing for 3 h to finish the

stabilization process; 2) in N2 atmosphere, heating up to 500°C at the rate of 2°C min−1

and

annealing for 1 h, then heating up to 1000°C at a rate of 5°C min−1

and annealing for 1 h.

CFMs from NCP (CFM-NCP), two-dimensional typically electrospun PAN fiber mat (2D-

ECFM) and layered NCP-PAN (layered-CFMs) were prepared for subsequent measurement.

6.2.2 Electrode preparation

All above CFM electrodes and including commercial carbon felt (CCF) (SGL Carbon GmbH)

were utilized as electrodes for the growth and investigation of (anodic) electrocatalytic biofilms.

They were cut into 1×2 cm2 pieces and were glued onto graphite foil paddles (serving as current

collector) using a conductive resin produced from two-component resin mixed with carbon

black particles.

6.2.3 Bioelectrochemical measurements

All electrochemical experiments were carried out as half-cell experiments under potentiostatic

control, using a three-electrode arrangement consisting of a working electrode, an Ag/AgCl

reference electrode (sat. KCl, 0.195 V vs. SHE) and a graphite plate counter electrode (size of

4×5 cm2). The experiments were conducted under control of a potentiostat (Autolab

PGSTAT30, equipped with six array channels) at 35°C (Patil, et al., 2010). All electrodes were

put in one chamber, a potential of 0.2 V (vs. Ag/AgCl) was applied on working electrode, and

the current was recorded in real time. To assure comparability and reproducibility up to six

different anode materials were measured simultaneously in one electrochemical cell.

The medium for bacterial growth was prepared with 10 mM Sodium acetate in 0.05 M sodium

phosphate buffer solution (pH 6.8). The bacterial communities were secondary biofilms which

were preselected from wastewater (wastewater treatment plant Steinhof, Braunschweig)

following procedures described in Ref. (Liu, et al., 2008). The electrochemical performance

tests were conducted when the biofilm activity reached stationary level.

6.2.4 SEM imaging

The fixation and drying of biofilm samples for morphology characterization were prepared as

follows: 1) fixed by 5 wt% glutaric aldehyde in 0.05 M phosphate buffer solution (pH=7.0); 2)

dehydrated in a graded series of aqueous ethanol solution; 3) then taken out and naturally dried

at room temperature. Scanning electronic microscopy (SEM) images were obtained from JSM-

7500F equipment under a voltage of 5 kV. The energy dispersive X-ray analysis (EDX) was

Page 106: PhD Thesis Alessandro Carmona2012

-85-

conducted by a CamScan SEM (Model S2-80DV). The mean pore size of the CFM samples was

measured by a capillary flow porometer (PMI, CFT-1200AEXL) using dry up/wet up method.

The ohmic resistance of carbon fiber mats was measured by stand four-point method using a

Keithley 2000 multimeter at room temperature.

6.3 Results and discussion

6.3.1 Properties and performance of carbon fiber mat electrode materials

Generally, NCPs in the form of tissue papers are made of cellulosic fiber pulp and are used in

daily life as toilet papers, paper handkerchief etc. The NCPs used in this work, were porous and

had a mean pore size of about 23 μm with layer thickness of about 20 μm, as shown in SEM

images (Fig. 6-1A and 6-1B). The resulting NCP-CFM exhibited a low electrical resistivity of

about 7.3 Ω cm. The properties of different CFMs are summarized in Table 6-1.

Table 6-1 Properties and anodic performance of carbon fiber mats.

Sample Mean pore

size/ μm

Porosity/

%

Resistivity/

Ω cm

Geometric current

density/ mA cm-2

CCF 47.0 95.7 0.2 1.21

NCP-CFM 38.0 93.6 7.3 0.53

2D-ECFM 0.6 90.0 8.0 0.17

Layered-CFM 2.3 98.5 2.0 2.00

The EDX spectrum shown in Fig. 6-1C indicated that the NCP-CFM contained a very small

amount of normal metal elements, e.g. Na, Al, K and Ca, in the form of phosphate or sulfate

salts. These elements were expected to show no adverse effects on the growth of

microorganisms.

Taking these features of NCPs into consideration, in this work, thin layers of NCP were selected

as support for LBL-electrospinning of PAN fibers to fabricate layered-CFM. In this layered-

CFM, the PAN fibers of about 500 nm diameter were assembled on the NCP (10–30 μm) layers

(Fig. 6-1D and 6-1E). The layered-CFM had a high porosity of 98.5% owing to a large gap of

over 50 μm between layers and small electrospun fibers diameter (Fig. 6-1E). This layered-CFM

structure as seen by SEM proved the hypothesis and would provide habitat for growth of layered

microbial biofilms.

The anodic performance of this layered-CFM was tested in a batch half-cell MFC. The

bioelectrocatalytic current generation curves were recorded versus time. They originated from

Page 107: PhD Thesis Alessandro Carmona2012

-86-

the oxidation of the substrate (acetate) catalyzed by microbial biofilms at the working electrode

according to the following equation:

eHCOOHCOOCHcatalysebioelectro

872 223 Equation 6-1

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.

Page 108: PhD Thesis Alessandro Carmona2012

-87-

6.3.2 Biocatalytic current generation at carbon fiber mat electrode materials

The bioelectrocatalytic current generation curves of five cycles are shown in Fig. 6-2. The

layered-CFM anode generated a maximum geometric current density of over 2.0 mA cm−2

. This

is higher (about 65%) than that obtained from CCF of 1.21 mA cm−2

and NCP-CFM with ten

layers of about 0.53 mA cm−2

.

The anodic current density from layered-CFM was ten times higher than that from 2D-ECFM of

only 0.17 mA cm−2

. The possible reasons for the high anodic current density from layered-CFM

are 1) the large space between layers provided room for high density biofilms growth and makes

the substrate supply into the layer easy, and 2) electrospun fiber layer with smaller diameter

induces microorganisms to form thick and stable layered-biofilms.

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.

6.3.3 Analysis of electroactive biofilms grown at carbon fiber mat electrode materials

with Scanning electron microscopy

To confirm the anodic performance, biofilms in the CFMs were investigated by scanning

electron microscope (SEM) (JSM-7500F). Thick and continuous layered biofilms were grown in

the layered-CFM, as shown in Fig. 6-3A to Fig. 6-3C. The biofilms in the first layer had a

thickness of around 10 μm (Fig. 6-3C), and it became thinner in the inner layers due to substrate

Page 109: PhD Thesis Alessandro Carmona2012

-88-

supply limitation. It revealed that the thickness of biofilms in the inner layers can be increased if

the gap between layers further increased for efficient nutrition transportation. Due to the big

pore size in the CCF resulted by big fiber diameter, the microorganisms were wrapped around

the big carbon fibers but did not form continuous layered-biofilm over the whole fiber mat (Fig.

6-3D and E).

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.

Page 110: PhD Thesis Alessandro Carmona2012

-89-

For the 2D-ECFM, as shown in Fig. 6-3F, only a very thin biofilm was grown on the surface of

2D-ECFM owing to the small pore size among fibers (Fig. 6-1F). The small pore size hindered

the microorganisms going inside and generated extremely low current density of only 0.17 mA

cm−2

. In summary, here it has been showed that small fiber diameter and proper pore size

combined with sufficient three dimensionality are essential features for the growth of thick and

continuous biofilms as well as for generation of high current densities.

6.4 Conclusions

Layered-CFM had been prepared and used for the anode in microbial fuel cells. These results

show that carbon materials with layered architecture and small fiber diameter are suitable for

layered biofilms growth with high cell density. Thick and continuous layered biofilms were

grown on this layered-CFM and generated high current density. This investigation also revealed

that if the gap between the layers within the layered-CFM can be further increased, thick layered

biofilms would grow in every layers of the entire layered-CFM and much larger current

densities would be obtained. The cellulose-based carbon fiber mat might provide a low cost and

highly efficient electrode for the anode in microbial fuel cells.

Page 111: PhD Thesis Alessandro Carmona2012

-90-

CHAPTER VII CHAPTER VII

7 Electroactive mixed culture derived biofilms in microbial

bioelectrochemical systems: the role of pH on biofilm

formation, performance and composition

7.1 Introduction

Electrochemically active microbial biofilms not only play a key role in environmental oxidation

reduction cycles, e.g. (Nielsen, et al., 2010), but also in microbial bioelectrochemical systems

(BES) (Rabaey, et al., 2010). Within this seminal technology microbial biofilms are exploited

for anodic oxidation reactions (Logan, 2009, Lovley, 2008b, Schröder, 2007) as well as cathodic

reduction reactions, e.g. (Harnisch and Schröder, 2010). These latter reactions may range from

the oxygen reduction in microbial fuel cells (MFC) to the reductive production and/ or

upgrading of chemicals, e.g. H2, in microbial electrolysers.

Common to the majority of these BES applications is the biofilm at the anode that is responsible

for the microbially assisted oxidation of the substrate (i.e. wastewater constituents). Except for

pure culture studies, which are highly relevant concerning the investigation of fundamentals, the

anodic biofilm in BES are generally formed from natural bacterial sources, i.e. inoculums, like

wastewater. The wastewater derived biofilms exploited in the initial phase of BES research

often possessed an only minor bioelectrocatalytic activity (Kim, et al., 2001) and consequently

different enrichment procedures were presented leading to an increased anodic biofilm

performance, see e.g. (Kim, et al., 2005, Liu, et al., 2008, Rabaey, et al., 2004).

Up to now, the majority of BES studies using mixed culture biofilms were performed using

laboratory conditions tailored towards highest activity, i.e. metabolic turnover, and thus

maximum current production. However, as BES technology has to be integrated into wastewater

treatment technology lines (Rozendal, et al., 2008) it has to be taken into account that the

biofilms may face different, often suboptimal and quickly varying abiotic conditions during their

formation and operation. This is especially a challenge when wastewater is used as feed, since

its quality changes quickly due to the amount and kind of the various inflow sources. Recently,

it has been demonstrated on the example of the operation temperature (Patil, et al., 2010) that

the influence of external environmental conditions can be severe.

Page 112: PhD Thesis Alessandro Carmona2012

-91-

Concerning the influence of the pH-value in the anodic compartment in BES, all recent studies

were restricted to a comparably narrow pH-window around pH neutral, e.g. (Biffinger, et al.,

2008, He, et al., 2008, Hong, et al., 2009, Jadhav and Ghangrekar, 2009, Liu, et al., 2005, Puig,

et al., 2010). Furthermore, acidophilic (Borole, et al., 2008) or alkalophilic (Liu, et al., 2010b)

microorganisms were exemplarily studied for a potential application of BES under extreme pH

conditions. Yet, as all these studies were performed in entire MFC devices, in which not only a

potential pH dependent biofilm performance contributes to the overall BES behaviour, but also

the pH-dependence of several technical operational parameters like that of the ion transfer

between anode and cathode (Harnisch and Schröder, 2009, Rozendal, et al., 2006a) or the

cathodic oxygen reduction reaction (Zhao, et al., 2006). Whereas the latter technological aspects

can be compensated by adequate technical measures (e.g. tailored geometries and materials), the

anodic biofilm may determine the overall pH-window of possible BES application. The aim of

this study is to provide information on the pH-influence of these biofilms from the short to

medium time frame (hours to days), as pH-associated metabolic adaptations take place within

minutes to hours (Siegumfeldt, et al., 2000).

Thus, in the present study the influence of the pH during formation and operation of natural

community derived anodic microbial biofilms was investigated using pH-values between pH 3

and pH 11. The biofilm formation, electrochemical performance, and redox-behaviour were

explored. Furthermore, the microbial structures and compositions of exemplary microbial

biofilms were analysed using flow-cytometry and terminal restriction fragment length

polymorphism (T-RFLP) analysis. The structures of the various upcoming communities in the

anode chambers were correlated with pH sensitivity, current production and biofilm formation

exploiting the Dalmatian-Plot and n-MDS similarity analysis.

7.2 Materials and methods

7.2.1 General conditions

All microbiological and electrochemical experiments were conducted under strictly anoxic

conditions at 35°C. If not stated otherwise, all reported pH-values in this study refer to the initial

growth medium pH within the electrochemical cell. All chemicals were of analytical or

biochemical grade. If not stated otherwise, all potentials provided in this article refer to the

Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE). All reported data are based on at least

three independent biological biofilm replicates and two replicates per biofilm for the continuous

flow-mode operation.

Page 113: PhD Thesis Alessandro Carmona2012

-92-

7.2.2 Electrochemical set-up

All electrochemical experiments were carried out under potentiostatic control, using three-

necked-flasks (250 mL) with three electrode arrangement consisting of the working electrode

(projected surface area; 8.00 cm2), a Ag/AgCl reference electrode (sat. KCl, Sensortechnik

Meinsberg, Germany, 0.195 V vs. SHE) and a counter electrode. The working and counter

electrodes used throughout this study were graphite rods (CP-Graphite GmbH, Germany). The

counter electrode was separated from the growth medium by a Nafion® 117 perfluorinated

membrane. The experiments were conducted with a Potentiostat/Galvanostat Model VMP3

(BioLogic Science Instruments, France), equipped with 12 independent potentiostat channels.

Cyclic voltammetry (CV) was performed during turnover and non-turnover conditions at a scan

rate of 1 mV s-1

in accordance with previous studies, e.g. (Fricke, et al., 2008, Srikanth, et al.,

2008). The current density is reported per projected surface area and denominated as “geometric

current density”.

7.2.3 Microbial inoculum and growth medium

The source for the microbial inoculum was primary wastewater collected from the WWTP

Steinhof, Braunschweig (Germany). The pH of the wastewater inoculum was 6.7±0.1 all time.

Always the identical inoculum was used for a consecutive set of experiments (see Section 7.3.3.)

The bacterial growth medium was prepared as reported by Kim et al. (Kim, et al., 2005). It

contained NH4Cl (0.31 g L−1

), KCl (0.13 g L−1

), NaH2PO4•H2O (2.69 g L−1), Na2HPO4 (4.33 g

L−1

), trace metal (12.5 mL) and vitamin (12.5 mL) solutions (Balch, et al., 1979). Acetate (10

mM) served as substrate in the growth medium. The pH values used during this study were 3, 5,

6, 7, 8, 9 and 11. The pH of the growth medium was adjusted to the desired value by using 1 N

NaOH or O-phosphoric acid. In the cathode chamber, buffer solutions were set to an equal pH-

value as the anodic pH and replenished in line with the anode solutions in the fed-batch

experiments. In order to ensure anaerobic conditions the growth medium was purged with

nitrogen at least for 20 min before use.

7.2.4 Biofilm growth (fed-batch experiments)

As described by Liu et al. (Liu, et al., 2008) for the formation of primary biofilms, 6.5 mL of

wastewater were inoculated into the sealed cell, containing 193 mL medium spiked with 10 mM

acetate as carbon and energy source. The cell was operated at 35°C. For consecutive cultivations

(that is exchanging the medium of the anode chamber by removing the old medium refilling it

Page 114: PhD Thesis Alessandro Carmona2012

-93-

with fresh medium and carbon source) 80% of the anode solution was exchanged, the volume

was chosen for practical reasons of reactor operation, but no new inoculation by a wastewater

took place. The exhausted substrate solutions of consecutive replenishing cycles were

denominated as “CX”, e.g. C1 to C5 for a 5 week operation period. During medium

replenishments, the cathode solution was also completely replenished using solutions of

identical pH like in the anode compartment. A constant potential of 0.2 V was applied to the

working electrode to facilitate the formation of a bioelectrocatalytic biofilm. The growth of the

biofilm was monitored by measuring the bioelectrocatalytic oxidation current. The exhausted

substrate was replenished regularly and the substrate level was monitored via HPLC. The data

for the maximum current generation at different constant pH-values (section 7.3.1.) are based on

biofilms grown for at least 4 fed-batch cycles and showing a constant performance (see e.g.

(Liu, et al., 2008)). These biofilms were also used for the flow-through (section 7.3.2.) and

cyclic voltammetric (7.3.3.) experiments. Furthermore selected fed-batch experimental runs

were used for the microbial analysis (7.3.4.).

7.2.5 Biomass determination

At least three independent samples per biofilm (each 1.2 mL) were spinned down at 17900 g for

10 min at 4°C in tubes, which prior to the analyses were dried at 105°C for 24 h. The

supernatant was removed and the procedure repeated until a cell pellet was accumulated (usually

less than 5 times). Subsequently the identical drying procedure was applied and the mass

difference of each tube, representing the dry mass per biofilm sample, was determined.

7.2.6 Metabolic analysis

Acetate consumption was analysed by HPLC (Spectrasystem P400, FINNIGAN Surveyor RI

Plus detector, Fisher Scientific, Germany) equipped with a Rezex HyperREZ XP Carbohydrate

H+ 8 µm column. Chromatograms were recorded at room temperature with 0.005 N sulphuric

acid as eluent.

7.2.7 Continuous flow mode operation and pH-regime studies

Two plastic tanks (10 L each) served as reservoirs for the substrate and buffer solutions. The

flow rates of both solutions were maintained at 0.5 mL min-1

using a peristaltic pump (IP 65,

ISMATEC, Laboratoriumstechnik GmbH, Germany). After adjustment of the biofilms to

continuous flow conditions – represented by establishing a continuous current generation for at

least 12 h - the biofilms were exposed to a pH-ramp from the initial pH-value to pH 5 and then

Page 115: PhD Thesis Alessandro Carmona2012

-94-

to alkaline pH up to pH 11 using a step wise pH decrease/increase with the interval of pH 1 by

changing the influent solution tanks. Thereby the steady-state current at every pH-value was

recorded.

7.2.8 Microbiological analysis

7.2.8.1 Flow-cytometry

Flow cytometry was used to resolve the community structure both in the anode surface and the

replenished substrate solutions on the single cell level. Therefore, every cell in the system was

measured according to the cells’ specific characteristics. These were morphological features

analysed by forward scattering (FSC) and related to cell size and DNA contents which were

specifically stained with the AT specific fluorescent dye DAPI. Every bacterial cell contains at

least one chromosome of a certain length and information. Some cells contain chromosomes of

different length and information whereas most cells contain several copies of them due to the

cells activity state in the cell cycle. After cytometric analysis of these parameters, cells cluster in

distinct patterns within so called dot plots. These patterns represent fingerprint like pattern of a

certain community structure. The patterns are very stable and can easily be reproduced (Müller

and Nebe-Von-Caron, 2010). Changes in community structures can quickly be visualized.

7.2.8.1.1 Sample fixation and DNA staining

Cells were harvested from the wastewater inoculum and from the anodic biofilms formed at pH

6.0, 7.0 and 9.0 and were conserved in fixation buffer (pH 7.0) with 10% sodium azide (Merck,

Germany) dissolved in PBS (1 ml fixation buffer for app. 3 x 108 cells ml-1

) for a maximum of 9

days. Aliquots of the fixed samples were washed twice in 2 ml PBS by centrifugation at 3200 x

g for 5 min and treated gently within an ultrasonic bath for 5 min to dissolve the biofilm. Two

mL of diluted cell suspension were treated with 1 mL solution A (2.1 g citric acid/0.5 g Tween

20 in 100 mL bidistilled water) for 10 min, washed and resuspended in 2 mL solution B (0.68

µM 4‘,6-diamidino-2‘-phenylindole (DAPI, SIGMA), 400 mM Na2HPO4, pH 7.0) and stained

for at least 60 min in the dark at 20°C.

7.2.8.1.2 Multiparametric flow-cytometry

Flow-cytometric measurements were carried out using a MoFlo cell sorter (DakoCytomation,

Fort Collins, CO, USA) equipped with two water-cooled argon-ion lasers (Innova 90C and

Innova 70C from Coherent, Santa Clara, CA, USA). Excitation by 400 mW at 488 nm was used

Page 116: PhD Thesis Alessandro Carmona2012

-95-

to analyse the forward scatter (FSC) and side scatter (SSC) as trigger signal at the first

observation point, using a neutral density filter with an optical density of 2.3. DAPI dye was

excited by 100 mW of ML-UV (333-365 nm) at the second observation point. The orthogonal

signal was first reflected by a beam-splitter and then recorded after reflection by a 555 nm long-

pass dichroic mirror, passage by a 505 nm short-pass dichroic mirror and a BP 488/10. DAPI

fluorescence was passed through a 450/65 band pass filter. Photomultiplier tubes were obtained

from Hamamatsu Photonics (models R928 and R3896; Hamamatsu City, Japan). Fluorescent

beads (Polybead Microspheres: diameter, 0.483 µm; flow check BB/Green compensation Kit,

Blue Alignment Grade, ref. 23520, Polyscience, USA) were used to align the MoFlo (coefficient

of variation – CV value - about 2%). Furthermore, an internal DAPI-stained bacterial cell

standard was introduced for tuning the device up to a CV value not higher than 6%. Cell

aggregation was not observed, thus clearly separated sub-communities were analyzed.

7.2.8.2 T-RFLP and Sequencing

T-RFLP gives information on phylogenetic relationships of bacteria and was therefore used to

prove the presence of certain bacteria on the anode biofilm and within the anode chamber

community. Sequencing was used to certainly affiliate the anode biofilm species to the data

base.

Fixed samples were spinned for 10 min at 17900 g at 4°C and the pellets stored at -20°C until

further analysis. DNA was extracted using a Chelex based method (Giraffa, et al., 2000).

Depending on the pellet size 150 or 300 μL 10% (w/v) Chelex were used. PCR, t-RFLP and

sequencing were performed as described earlier (Harnisch, et al., 2011). The restriction

endonucleases RsaI and MspI (New England Biolabs, Schwalbach, Germany) were used with

the corresponding buffer. Partial sequencing of the 16S rRNA gene was performed with the

primers 27f und 519r and the sequences deposited in the GenBank database under accession

numbers JN393007–JN393010.

The lengths of the fluorescent terminal restriction fragments (T-RFs) in the range from 50 to 600

bp were determined with the genemapper V3.7 software (Applied Biosystems, Weiterstadt,

Germany). Data normalization was performed with an R implementation based on Abdo et al.

(Abdo, et al., 2006) and statistical analysis was done with PAST

(http://folk.uio.no/ohammer/past/).

Page 117: PhD Thesis Alessandro Carmona2012

-96-

7.3 Results and discussion

7.3.1 Biofilm formation and performance at different constant pH

Figure 7-1 summarizes the results of potentiostatic fed-batch experiments of primary,

wastewater derived, biofilms (Liu, et al., 2008, Patil, et al., 2010) for different pH-values during

biofilm formation and operation. It depicts the maximum geometric current densities as well as

coulombic efficiencies of mature primary biofilms grown and operated at different pH-values.

Thereby, Figure 7-1 clearly shows that only at pH 7 a high average current density of 821 µA

cm-2

and coulombic efficiency of 82% was achieved, whereas more acidic or more alkaline

conditions, i.e. pH-values differing from the pH of the municipal wastewater (pH 6.7) that

served as inoculum, resulted in a clear decrease of current density and coulombic efficiency.

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.

One can clearly see that at extreme pH-values, i.e. at pH 3 and pH 11, no bioelectrocatalytic

activity was established, whereas the activity at pH neutral is in accordance with previous

studies (e.g., (Patil, et al., 2010)). This result was not unexpected, as the primary wastewater

Page 118: PhD Thesis Alessandro Carmona2012

-97-

from the local treatment plant possessed an almost neutral pH, and thus the microbial

communities therein can be assumed to be properly adapted to this pH-environment.

Interestingly, whereas the average maximum geometric current density, as a measure of the

maximum performance, is only slightly decreasing from pH 7 to 9 (~10%) the coulombic

efficiency, representing the cumulative performance, is more severely affected. It decreases

from 82% at pH 7 via 73% at pH 8 to 39% at pH 9. These results clearly show that – when using

identical inoculums of pH neutral wastewater– electrocatalytic active biofilms can be derived

only in a limited pH-window (here from pH 6 to pH 9).

In conclusion it can be stated that the more the pH-value during biofilm formation and operation

deviates from the pH of the bacterial source (pH neutral wastewater), the lower and less efficient

its bioelectrocatalytic activity becomes.

7.3.2 Biofilm performance at varying pH-environment during operation

Subsequently, the performance response of well developed biofilms on the variation of the pH-

environments was assessed, in order to mimic the influence of a changing pH in the wastewater

influent. This is not only highly relevant concerning the technical applicability of the

electroactive biofilms, but can furthermore be regarded as stress-test from the microbiologist’s

perspective. Figure 7-2A shows the bioelectrocatalytic current production of a mature, i.e.

constant current producing, biofilm (grown at pH 7) in a continuous flow mode reactor (see

7.2.7.) exposed to varying pH-values.

One can clearly see that the bioelectrocatalytic performance declines when exposing the biofilm

to more acidic conditions. For the acidic pH-environment the bioelectrocatalytic performance

drops almost completely down, from about 800 µA cm-2

at pH 7 to less than 40 µA cm-2

at pH

5. Remarkably, as Figure 7-2 A shows, the complete bioelectrocatalytic activity is re-established

within less than 24 h, when switching the pH back to pH 7. Furthermore, Figure 7-2 A shows

that an exposure to more alkaline pH first slightly increases the bioelectrocatalytic current

production. However, longer exposure times and especially highly alkaline conditions lead to an

irreversible biofilm degradation that cannot be re-established when the biofilm is exposed to pH

7 again (see Figure 7-2A).

This biofilm degradation often went along with a biofilm detachment from the electrode surface,

as can already be seen by visual inspection (see Figure S11-11 in Supplementary information).

Page 119: PhD Thesis Alessandro Carmona2012

-98-

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)).

Page 120: PhD Thesis Alessandro Carmona2012

-99-

Figure 7-2B summarises the results of similar experiments, as in Figure 7-2A, performed with

biofilms grown at pH 7 and pH 8 , by depicting the respective current density as function of the

applied pH-value. Commonly, the operational window is limited to pH-values between pH 6

and pH 9, which is well in accordance with the pH-window found for the formation of

respective bioelectrocatalytic biofilms from wastewater inoculums (see Figure 7-1).

Furthermore, it can be concluded that pH 7 grown biofilms (showing 360 µA cm-2

) are about

twice as active at pH 6 than biofilms formed at pH 8 (171 µA cm-2

). Thus, one can assume that

these biofilms are better adapted to the respective higher proton concentration. Furthermore,

these results are well in accordance with preceding MFC studies, in which a reversible

adaptation of anodic biofilms to varying pH-conditions, resulting in different reactor

performances, was demonstrated, e.g. (Jadhav and Ghangrekar, 2009).

7.3.3 Influence of the pH and buffer capacity on the electron transfer

In order to elucidate the influence of the anode chamber’s pH and ionic strength/ buffer capacity

on the electron transfer cyclic voltametric measurements (CV) were performed. Consequently,

to minimize the impact of any biological variability in the experiments always identical biofilms

were studied for varying pH-conditions by changing the medium in the anode chamber between

the experiments (overall duration was less than 6 h). Figure 7-3 shows the non-turnover, i.e.

acetate depleted, CVs of a pH 7 grown biofilms at different pH-values in pure electrolyte

solutions.

The correlation between pH-value and redox-potentials of the active sites depicts that with

decreasing pH the formal potentials of all redox-active moieties are shifted towards more

positive values. The redox-centres, most likely related to the direct electron transfer sites of

Geobacter (Fricke, et al., 2008, Liu, et al., 2008) as these are the dominating microorganism in

this biofilm (see below) are both ascribed to c-type cytochromes (Millo, et al., 2011), shifting

more than 140 mV from pH 9 to pH 6. This potential shift of about 47 mV/ pH is well in

accordance with previous studies for G. sulfurreducens (that was a dominant microorganism in

our biofilms, see 7.3.5.) on glassy carbon electrodes (Katuri, et al., 2010) and acetate derived

biofilms (Yuan, et al., 2011), both studied for pH 6 to pH 8. Interestingly, when biofilms were

exposed to pH 5, which after a longer exposure was generally leading to a complete biofilm

detachment, no constant and distinctive CV-curve could be recorded.

Page 121: PhD Thesis Alessandro Carmona2012

-100-

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).

Analysing both oxidation peaks of the direct electron transfer proteins showed a lower

discrimination between the redox-couples at more acidic conditions (data not shown). This

finding, indicating a different pH-dependence of both direct electron transfer sites, needs a more

detailed analysis applying highly-sensitive electroanalytical methods as well as hyphenated

techniques, e.g. spectroelectrochemical approaches, e.g. (Millo, et al., 2011).

Interestingly, the more positive oxidation peak, located at about -100 mV at pH 7, which is not

involved in the bioelectrocatalysis (Fricke, et al., 2008), shows also a pH dependence.

Subsequently, in order to elucidate if the demonstrated dependence of the electron transfer is

purely associated to H+-transfer or depends on a charge balancing counter ion transfer in

Page 122: PhD Thesis Alessandro Carmona2012

-101-

general, CVs for constant pH but varying buffer capacity were recorded (Figure S11-1A). These

show clearly that a variation of the buffer capacity has almost no influence on the formal

potentials of the active site (Figure S11-1A). Only a decreasing CV-resolution, which might be

attributed to the ohmic resistance (i×R-drop) in the biofilm, can be detected. Thus, the buffer

capacity (and ionic strength) has no influence on the electron transfer thermodynamics.

In contrast, the buffer capacity determines the maximum current density for turnover conditions,

i.e. the maximum bioelectrocatalytic performance. An increase by one order of magnitude in

buffer concentration caused an increase of the maximum current production of about 40% for

the identical biofilm (see Figure S11-1B: 250 µA cm-2

and 400 µA cm-2

were achieved for 5

mM and 50 mM buffer concentration, respectively).

This finding is well in accordance with previous studies showing the pH gradient within a

biofilm from the electrode surface to the electrolyte solution is severely limiting the

bioelectrocatalytic performance (Torres, et al., 2008).

These results clearly reveal that the charge balancing ion (proton) transfer through the biofilm

represents a severe bottleneck of the electrocatalytic biofilm activity. This was already indicated

in prior CV-studies showing a differing mass-transfer dependence of these biofilms for low and

high scan rates (Fricke, et al., 2008, Srikanth, et al., 2008) and recent results mapping the pH-

gradient within G. sulfurreducens biofilms (Franks, et al., 2009).

7.3.4 Microbial biofilm analysis

In order to elucidate the microbiological reasons for the variations in the bioelectrocatalytic

performances of the natural community derived biofilms gained at different pH-values, flow-

cytometric and T-RFLP-analyses including 16S rRNA gene sequencing were performed. In

total, two out of several parallels, in the following denominated as electrode-set 1 and electrode-

set 2, were investigated on their microbial structure (by flow-cytometry) and composition (by T-

RFLP) at pH 6, pH 7 and pH 9 using the identical inocula of pH 6.7 wastewater for the

respective parallels and acetate as carbon and energy source.

Figure 7-4 shows, on the example of a pH 7 derived biofilm at electrode-set 1, the flow-

cytometric analysis of a bacterial anode chamber community after wastewater inoculation. Five

Page 123: PhD Thesis Alessandro Carmona2012

-102-

successive fed-batch medium exchanges were performed (denominated as C1 to C5, see

materials and methods) and the dynamics of the planktonic anode chamber community followed

until final development of the active current producing anodic biofilm. The resulting datasets

showed the clustering of cells of the community to distinct sub-communities (Müller and Nebe-

Von-Caron, 2010). Presence and absence as well as the relative abundances of cell numbers

within these clusters gave a fingerprint like information on the structure of the microbial

community. It is obvious that the structure was changing over the five feeding cycles C1 to C5

which is due to the adaptation of the community structure from complex wastewater to acetate

as sole carbon and energy source. It shows that the microbial community responded sensitively

to changes in its microenvironments (Günther, et al., 2011).

Additionally, the cytometrically determined structures of the communities in the anode chamber

during the different feeding cycles differed strongly from that of the inoculum (see Figure 7-4).

Despite the miscellaneous structure variation in the chamber broth (flow-patterns C1 to C5), a

microbial biofilm evolved at the anode dominated by mainly one phylotype.

For both electrode-sets a similar maximum current density and columbic efficiency for biofilms

formed at pH 7 was achieved, with in average jmax=740 µA cm-2

and CE=99.8%. The anode

biofilms of both electrode-sets were dominated by one phylotype, as can be seen from the

respective flow-cytometric analysis. The related T-RFLP chromatograms for restriction

digestion analysis displayed only one dominant peak at 238 bp (92% of the total peak area for

pH 7) (see S11-2). The subsequent 16S rRNA PCR products of the two investigated pH 7 anode

biofilms were partially sequenced and resulted in the identification of the genus Geobacter using

the RDP classifier. The maximum score in the BLAST search, excluding

uncultured/environmental samples, resulted in the identification of Geobacter sulfurreducens

(CP002031.1) with a maximum identity of 97%. Comparison of these sequences with previous

results in the work group shows a 100% identity (Harnisch, et al., 2011). This dominance of

Geobacter sulfurreducens for the respective conditions is well in line with a previous study

(Torres, et al., 2010).

Page 124: PhD Thesis Alessandro Carmona2012

-103-

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.

As was shown for both of the electrode-sets and for the three different pH-values investigated

the community structure responded with rapid and dynamic changes in community structure

since it adapted easily to the varying abiotic conditions. To align fingerprints originating from

‘healthy’ and active biofilm launching communities apart from those connected with inactive

biofilms a similarity analysis was performed on the basis of the cytometric dot plot clusters. The

approach divides the productive from the non-active communities in the sense of their potential

to establish active biofilms with high current production efficiency. Thus, cytometric analysis

gives information on the potential ability of a community to set up active biofilms. The

approach is quickly performed and cheap. This is a huge advantage in comparison to

phylogenetic approaches like T-RFLP and sequencing techniques which are considerably more

cost and time intensive.

Page 125: PhD Thesis Alessandro Carmona2012

-104-

The n-MDS (see Section 11.9 in Appendix C) analysis of the anode chamber microbiology

revealed that the planktonic communities and the biofilms are changing gradually in their

structure and dynamics depending on experimental conditions. The pH-environment seems to

influence the planktonic community structure more distinct than the consecutive acetate fed-

cycle conditions since all of the latter are more or less closely clustered (high similarity). This

finding points towards a high stability of the planktonic microbial community over longer time

periods (more than 2 weeks) under stable pH and substrate (acetate) conditions. However,

changes in the pH caused also changes in the planktonic community structure, clearly shown by

the separated respective Dalmatian plots. The related consecutive fed-cycles at the various pH-

values did not cluster together (low similarity). Evaluating the anode biofilm patterns at pH 6,

pH 7 and pH 9 pronounced differences in the Dalmatian pattern were identified, resulting in a

clear separation from all planktonic community plots (low similarity).

Furthermore, high performing biofilms, identified to contain mainly Geobacter sulfurreducens

clustered together in the lower right corner of the n-MDS plot (electrode-sets 1 and 2, pH 7;

electrode-set 2, pH 6; electrode-set 1, pH 9, high similarity). The patterns of the low performing

electrodes (electrode-set 2, pH 9; electrode-set 1, pH 6) showed a different fingerprint and

clustered apart from the other electrodes but also from the planktonic communities (low

similarity).

The opposite development of high performing biofilms at pH 6 and pH 9 can be explained by

the different inocula used for the two electrode-sets 1 and 2. The different inocula were obtained

during either a summer (August) and winter (November) period from the wastewater treatment

plant and influenced the biofilm establishment insofar that either pH 6 (electrode-set 1) or pH 9

(electrode-set 2) resulted in a microbial underdevelopment represented by an only low

bioelectrocatalytic activity.

The two pH 7 biofilms showed a constant high bioelectrocatalytic activity and typical

population patterns for G. sulfurreducens, as measured by flow-cytometry. The further two high

performing biofilms found for pH 6 and pH 9 (with performance values of jmax=705 µA cm-2

and CE=50.2% for pH 9 (electrode-set 1) and jmax =191 µA cm-2

and CE=72.1% for pH 6

(electrode-set 2)) showed similar population patterns to the pH 7 grown biofilms (see Figure 7-

5). Here, the T-RFLP data presented the respective peak at 238 bp (84% of the total peak area

for pH 6 grown biofilms, Fig S11-4 (electrode-set 2); for pH 9 grown biofilms, S11-3

(electrode-set 1)) which also confirmed G. sulfurreducens using partially sequencing.

Page 126: PhD Thesis Alessandro Carmona2012

-105-

In contrast, when the biofilms possessed only a lower electrochemical activity degenerated

cytometric biofilm patterns were observed (more distant clustering in Figure 7-5); jmax=137 µA

cm-2

and CE=21% for pH 6 (electrode set 1) and jmax=0.27 µA cm-2

and CE=0.1% for pH 9

(electrode set 2). For the latter, also a decrease in anode dry biomass from 29.8 mg ± 0.2 mg (pH

7) to 9.8 mg ± 0.2 mg (pH 9) was detected. In both cases of low-performance biofilms G.

sufurreducens was not the dominating organism of the respective anode community (see S11-3

(pH 9, electrode set 2) and S11-4 (pH 6, electrode set 1)).

Thus, since substrate and electrode potential were identical for all experiments it is obvious that

the pH-environment as well as the microbial source were the strongest driving forces for

variations within the bioelectrocatalytic activity on the anode and the structures of the associated

planktonic microbial communities. These findings suggest that current production and efficiency

depend on a stable anode biofilm formation, allowing (for the applied conditions in this study)

the dominant growth of G. sufurreducens. The varying biofilm formation efficiency at different

pH-values is not surprising since G. sulfurreducens is known to possess its maximum growth

rate at pH 7 that is significantly lowered, e.g. at pH 6 (Franks, et al., 2009) - which is in

accordance with the DNA/FSC growth pattern detected via flow-cytometry.

Wastewater is highly variable in its composition and characterized by inconstant abiotic

parameters. How sensitive communities react to extrinsic parameters like wastewater

composition and temperature is shown within the Dalmatian plot with the two inocula clustering

apart from each other in their community pattern but also apart from the newly developed

communities in the anode chamber now using only acetate as substrate. Therefore it can be

assumed that wastewater composition and thus its microbial community severely influences the

ability to form a high performance biofilm at the respective pH-value other than the growth

optimum, as the environmental conditions and their variability (frequency and amplitude) define

which microbial species can establish and persist (Günther, et al., 2011). Furthermore, it has to

be pointed out that no other single microbial species dominated the low performance biofilms,

which in contrast where highly diverse. Here, different, complex ecological mechanisms,

including ‘priority effects’ and the formation of repellent EPS substances, may avoid the

dominance of a distinct phylotype for the respective operating conditions (Flemming and

Wingender, 2010, Van Gremberghe, et al., 2009).

Page 127: PhD Thesis Alessandro Carmona2012

-106-

Although T-RFLP pattern suggested a complex suspension community in the anode

compartment of all biofilms, there was seemingly no other bioelectrocatalytic microorganism

present that could take advantage and dominate the biofilm instead of Geobacter sulfurreducens.

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).

Page 128: PhD Thesis Alessandro Carmona2012

-107-

7.4 Conclusions

It is demonstrated that the pH-value plays an important role for the biofilm formation,

composition and performance of electroactive biofilms. Starting from pH-neutral wastewater as

inoculum, high performance biofilms were dominated by G. sulfurreducens, whereas a lower

performance went along with a higher microbial diversity. Analysing the impact of the pH-value

and buffer capacity/ ionic strength on identical biofilms shows that the latter influence the

maximum achievable current density, but not the formal potentials of the electron transfer

proteins. These, however, show a strong dependence on the pH-value of the solution - calling

for the further investigation of this phenomenon.

Page 129: PhD Thesis Alessandro Carmona2012

-108-

CHAPTER VIII

8 Electroactive mixed culture derived biofilms in microbial

bioelectrochemical systems: the role of inoculum and

substrate on biofilm formation and performance

8.1 Introduction

Bioelectrochemical systems (BESs) are a group of developing and promising technologies

targeting different kind of goals (Rabaey and Rozendal, 2010), from the production of

bioelectricity, via the production of biofuels (e.g., H2), to the production of valuable

biochemicals (e.g., H2O2). As seen in chapter 1, depending on the BES’s application (see Fig. 1-

6), a plenitude of applications can be conceived regarding the overall configuration of the BES

(Logan, et al., 2008, Logan, et al., 2006), from the membrane specificity (Harnisch and

Schröder, 2009) to the type of (bio) catalyst interacting at both electrodes (Franks, et al., 2010,

Rosenbaum, et al., 2011). BESs utilize the energy available in bio-convertible substrates via the

catalytic activity of electrochemically active biofilms developed at the electrode material (in this

case the anode). These biofilms are composed of a network of bacteria layers growing on the

electrode material that oxidize a substrate to finally transfer the harvested electrons to the anode

that serves as a microbial electron acceptor (Lovley, 2011).

Commonly in BESs the focus in using pure cultures of bacteria as bio-catalyst is the study of

fundamental phenomena such as the thermodynamic processes involved in microbial electron

transfer (see Chapter 2, 3 and 4). However most BESs take advantage of mixed culture derived

anodic biofilms due to their practical application. To find the optimal conditions for the

formation and performance of electroactive biofilms, different microbial sources and type of

substrates should be explored to get a more precise idea of the dynamics of the electrode

bacterial communities (Logan and Regan, 2006a, Pant, et al., 2010). It has been demonstrated

that many different factors influence both, the composition of the bacterial community in

electroactive biofilms in BESs and their performance (Franks, et al., 2010). Some of the studied

factors are, among others: temperature (Patil, et al., 2010), external ohmic resistance (Zhang, et

al., 2011), oxygen limitation (Biffinger, et al., 2009), flow rate (Ieropoulos, et al., 2010), pH

Page 130: PhD Thesis Alessandro Carmona2012

-109-

environment (see Chapter 7), type of inoculum and type of substrate as it has been explored in

this study.

Regarding the influence of the inoculum and substrate on the formation and performance of

anodic biofilms, one can find in the literature the following representative examples exploring

the influence of certain inocula and substrates. Min, et al. (Min, et al., 2005) compared two

types of inocula for power production: a pure culture of Geobacter metallireducens and a mixed

culture enriched from wastewater that commonly leads to the formation of a biofilm dominated

by a bioelectroactive bacteria, strain of the Geobacteraceae family (Harnisch, et al., 2011).

Since both biofilms were possibly conformed by similar electroactive bacteria is not surprising

that no significant difference was found in the performance between used inocula.

A similar approach was used by Ieropoulos, et al. (Ieropoulos, et al., 2010). In their research

they tested two inocula representative of complex communities of microflora found in the final

stages of wastewater treatment (anaerobic and aerobic effluent), an environmental inoculum

such as river water and a pure culture of a bioelectroactive bacteria such as G. sulfurreducens.

The study by Ieropoulos et al. showed no significant difference in the performance no matter

what inoculum was used. Furthermore although they report the characterization of the anodic

biofilm, no quatitative data were presented to allow a proper comparison. On the other hand,

Nimje et al. (Nimje, et al., 2012) tested two different inocula (wastewater and a pure culture of

Shewanella oneidensis MR-1) and four types of wastewaters as substrate sources (agriculture,

domestic, paper and food/dairy). Their study produced results which corroborated the findings

of a great deal of the previous BES experiments using different kind of inocula and substrates,

i.e., there was no significance difference in the performance of the tested systems. This probably

due to the mixture of a pure culture such as S. oneidensis MR-1 with wastewater as inoculum,

which probably led to the dominance of electroactive bacteria present in the wastewater and thus

masking the effect of S. oneidensis MR-1.

Additionally, there are a few studies focused on the influence of different substrates on the

biofilm formation. Velasquez-Orta et al. (Velasquez-Orta, et al., 2009) tested two kind of algae

as substrate for BESs. They demonstrated that in principle algae can be used as a renewable

source of electricity production. However they could not find significance differences by

feeding different kind of algae. From the different substrates used in comparative BES studies,

carbohydrates are the most used due to the preference of some electroactive bacteria for these

compounds (e.g., acetate in the case of Geobacter and lactate in the case of Shewanella). Several

Page 131: PhD Thesis Alessandro Carmona2012

-110-

research groups have studied the influence of some carbohydrates. For example Min and Logan

(Min and Logan, 2004) studied the influence of five specific substrates (glucose, acetate,

butyrate, dextran and starch) finding that when the system was fed with acetate the power

generation was sustained at high rates. In a similar study performed by Thygesen et al.

(Thygesen, et al., 2009) several BESs were fed with acetate, glucose or xylose as substrates.

Acetate produced the highest current probably due to a simpler metabolism than with glucose or

xylose. In another major study, Lee et al. (Lee, et al., 2008) quantified the impact of using

acetate and glucose as substrates on several experimental variables such as current and biomass

production, among others. The energy-conversion efficiency was significant higher with acetate

than with glucose. They attributed this to very low energy-conversion efficiency for glucose due

to a large increase of the anode potential. Additional analysis of the biomass on the anode

showed that although glucose allowed higher biomass density, it had a very low current density,

which supported the fact that the density of electroactive bacteria was very low.

Although acetate seems to be the best substrate for electroactive biofilms enriched from

wastewater samples, some recent studies show the contrary. For instance Cao et al. (Cao, et al.,

2010) demonstrated that by using three specific substrates like glucose, acetate and ethanol for

the growth of electroactive biofilms glucose is utilized in a more efficient way to produce

current than the rest of substrates. Results presented by Cao et al. (Cao, et al., 2010) were in

agreement with similar studies published by Sharma and Li (Sharma and Li, 2010) showing the

same trend in the utilization of different substrates by electroactive bacteria.

In a different category, experiments using a co-culture of bacteria which benefit from their

interaction should be mentioned because this type of studies allow us to understand the

ecological relationships of the microbiota in BESs, a necessary requisite to gain deeper insight

into their performance. For instance, Venkataraman and co-workers (Venkataraman, et al.,

2011) showed that the fermentation product 2,3-butanediol stimulates mutually beneficial

interactions between Pseudomonas aeruginosa PA14 and Enterobacter aerogenes in a BES

with glucose as the initial substrate under microaerobic conditions. They found that current

density by a co-culture of P. aeruginosa and E. aerogenes increased at least 14-fold compared to

the current density by either of these two bacteria alone; and that E. aerogenes fermented

glucose principally to 2,3-butanediol, which was subsequently consumed by P. aeruginosa. The

current production by a pure culture of P. aeruginosa with 2,3-butanediol was increased 2-fold

compared with glucose as the carbon source. This was due to enhanced phenazine production by

P. aeruginosa. Their study was the first to demonstrate metabolite based ‘‘inter-species

Page 132: PhD Thesis Alessandro Carmona2012

-111-

communication’’ in BESs, resulting in enhanced electrochemical activity. It also explains how

an inconsequential fermenter can become an important electrode respiring bacterium within an

ecological network at the anode.

As one can see, it exists a vast amount of BES studies using all kind of inocula and substrates,

however there is a lack of unifying studies that compare different inocula and substrates under

the same experimental conditions. Thus, in order to exclude the influence of operational

variables and to investigate only the effect of individual microbial inoculum source with sodium

acetate or sodium lactate as substrates, the experiments here presented were conducted with

half-cell set-ups under potentiostatic control (Fig. 8-1) investigating the general

bioelectrocatalytic activity (current density) and the voltammetric behavior.

8.2 Materials and methods

8.2.1 General conditions

All microbiological and electrochemical experiments were conducted under strictly anoxic

conditions at 35°C. All chemicals were of analytical or biochemical grade. If not stated

otherwise, all potentials provided in this article refer to the Ag/AgCl reference electrode (sat.

KCl, 0.195 V vs. SHE). All data are based on experiments during at least 5 semi-batch cycles of

2 independent biofilm replicates, and the standard deviations are presented in Fig. 8-2.

8.2.2 Electrochemical set-up

All electrochemical experiments were carried out under potentiostatic control, using three-

necked-flasks (250 mL) with three electrode arrangement consisting of the working electrode

(projected surface area; 8.00 cm2), a Ag/AgCl reference electrode (sat. KCl, Sensortechnik

Meinsberg, Germany, 0.195 V vs. SHE) and a counter electrode. The working and counter

electrodes used throughout this study were graphite rods (CP-Graphite GmbH, Germany)

contained in the same chamber (Fig. 8-1A). The experiments were conducted with a

Potentiostat/Galvanostat Model VMP3 (BioLogic Science Instruments, France), equipped with

12 independent potentiostat channels. The current density is reported per projected surface area

and denominated as “geometric current density”.

Page 133: PhD Thesis Alessandro Carmona2012

-112-

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).

8.2.3 Microbial inoculum and growth medium

There were four types of wastewater collected from the Waste Water Treatment Plant Steinhof,

Braunschweig (Germany) that served as the source for the microbial inoculum, i.e.: primary

wastewater, activated sludge, primary sludge and secondary sludge. Always the identical

inoculum was used for a consecutive set of experiments. The bacterial growth medium was

prepared as reported by Kim et al. (Kim, et al., 2005). It contained NH4Cl (0.31 g L−1

), KCl

(0.13 g L−1

), NaH2PO4•H2O (2.69 g L−1

), Na2HPO4 (4.33 g L−1

), trace metal (12.5 mL) and

vitamin (12.5 mL) solutions (Balch, et al., 1979). Sodium acetate (10 mM) or Sodium lactate

(10 mM) served as substrates in the growth medium. In order to ensure anaerobic conditions the

growth medium was purged with nitrogen for 30 min before use.

8.2.4 Biofilm growth in bioelectrochemical half-cells

The biofilm formation procedure was followed as described by Liu et al (Liu, et al., 2008) in

fed-batch experiments. For the biofilm formation 10 mL of individual microbial sample was

inoculated into the sealed electrochemical cell that contained 200 mL of the stirred growth

medium with substrate under study. A constant potential of +0.2 V was applied to the working

electrode to facilitate the biofilm formation. The biofilm growth was monitored by measuring

Page 134: PhD Thesis Alessandro Carmona2012

-113-

the bioelectrocatalytic oxidation current. For the initial (usually three) batch cycles, microbial

inoculum was added to the medium.

8.2.5 Cyclic voltammetry

Cyclic voltammetry (CV) was performed during turnover and non-turnover conditions in

accordance with previous studies, e.g. (Fricke, et al., 2008, Srikanth, et al., 2008). Potentials

were applied from -500 to +300 mV (vs. Ag/AgCl) at a scan rate of 1 mV s-1

with continuous

monitoring of the current response.

8.2.6 Metabolic analysis for coulombic efficiency calculation

Acetate and lactate consumption was analysed by HPLC (Spectrasystem P400, FINNIGAN

Surveyor RI Plus detector, Fisher Scientific, Germany) equipped with a Rezex HyperREZ XP

Carbohydrate H+ 8 µm column. Chromatograms were recorded at room temperature with 0.005

N sulphuric acid as eluent. The total coulombic efficiency (CE) was calculated by integrating

the current over time according to the method described by Logan et al. (Logan, et al., 2006).

8.3 Results and discussion

8.3.1 Current density production of enriched microbial electroactive biofilms as a

function of microbial inoculum and substrate

A significant difference in current generation was observed for all bioelectrochemical set-ups

(Fig. 8-2). However only visible biofilms were detected after 5 semi-batch cycles for

experiments using primary wastewater as inoculum (see Fig. 8-1B). As shown in Fig. 8-2, the

acetate-fed-reactor with primary waste water inoculum showed the highest current density (558

± 27 μA cm-2

, CE = 94 ± 1%), followed by lactate-fed-reactor with primary waste water

inoculum (460 ± 54 μA cm-2

, CE = 25 ± 12%). Secondary sludge resulted in the lowest current

outputs with both substrates in comparison to other inocula. The most possible reason for the

low current densities with secondary sludge as an inoculum could be the absence of high-

current-producing exoelectrogenic microorganisms to develop biofilms either through

competition with other microbes or an inability to use this specific substrate (Lee, et al., 2008).

Page 135: PhD Thesis Alessandro Carmona2012

-114-

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.

Activated sludge as inoculum although did not show as good performance as primary

wastewater, it was the second best current density producer amongst other microbial inocula

with 94 ± 3 μA cm-2

for acetate and for 165 ± 31 μA cm-2

lactate. Furthermore, the high

performance with primary wastewater for the formation of bioelectroactive biofilms

demonstrated its ability as efficient microbial inoculum source. The better performance with

primary waste water also indicated selective enrichment of electrocatalytically active microbes

on the anode and thus proved to be better candidates for the formation of mixed culture based

electroactive bacteria (Harnisch, et al., 2011).

Just two of the used microbial inocula showed significant current density production (primary

wastewater and activated sludge). This could be attributed to the complexity of these mixed

culture inocula (Angenent, et al., 2004, Logan and Regan, 2006a). As shown here and in

accordance with previous results (Chae, et al., 2009, Jung and Regan, 2007, Liu, et al., 2004,

Pant, et al., 2010), acetate was the preferred substrate for electricity generation with different

inocula in MFCs.

Page 136: PhD Thesis Alessandro Carmona2012

-115-

The low performance with primary and secondary sludge might be attributed to a low extent of

bacterial adhesion to the anode which is necessary for better performance. It has been shown

that electroactive biofilm formation at the anodes is an important factor to increase the current

production (Jiang, et al.). Furthermore, these microbial inocula contain a variety of non-

electrogenic bacteria that compete with electrogenic bacteria for the growth, which probably

slowed down the electroactive biofilm formation process and thus the overall bioelectrocatalytic

performance (Wagner, et al., 2002). Interestingly, Activated sludge exhibited better performance

with lactate than with acetate which might be because of involvement of lactate utilizing

microorganisms in this inoculum (Liu, et al., 2007).

8.3.2 Bioelectrocatalytic activity of enriched microbial electroactive biofilms as a

function of microbial inoculum and substrate

Cyclic voltammetry was performed for all established bioelectroactive biofilms formed from

four different inocula and fed either with sodium acetate (Fig. 8-3) or with sodium lactate (Fig.

8-4). Cyclic voltammograms during non-turnover (A, C, E and G in Fig. 8-3 and 8-4) and

turnover (B, D, F and H in Fig. 8-3 and 8-4) conditions with all set-ups confirmed the biofilm

associated current generation. Exemplary CVs of primary wastewater, activated sludge, primary

sludge and secondary sludge based electroactive biofilms indicated the different electro-

chemical behaviour with both substrates. As pointed out in section 8.3.1, only visible mature

biofilms were detected with primary wastewater as inoculum (see Fig. 8-1B). Maturity of

biofilms was confirmed from a constant maximum of current density production and a non-

changing CV shape after the third semi-batch cycle. For turnover CVs of biofilms enriched from

primary wastewater the formal potential of the active site (bioelectrocatalysis) was about -282

mV vs. Ag/ AgCl for sodium lactate fed biofilms and -248 mV for sodium acetate fed biofilms

(derived from the first derivative of CVs for turnover conditions in Fig. 8.5). This clearly

indicates that the used inocula considerably influenced the enrichment of electrochemically

active bacteria with different substrates.

Page 137: PhD Thesis Alessandro Carmona2012

-116-

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

.

Furthermore, the CV patterns during non-turnover conditions with all inocula (A, C, E and G in

Fig. 8-3 and 8-4) showed very different and complex redox behaviour as well and thus electron

transfer thermodynamics. After the third semi-batch cycle only biofilms enriched from primary

wastewater showed a non-changing CV shape (Fig. 8-3 and 4A). For these non-turnover CVs

the formal potential of the active site (bioelectrocatalysis) was about -300 mV vs. Ag/ AgCl in

agreement with previous results with electrodes modified with G. sulfurreducens biofilms

(Fricke, et al., 2008). This clearly indicates that the used inoculum considerably influenced the

enrichment of electrochemically active bacteria with different substrates. Furthermore, this also

demonstrates that the used conditions lead to the enrichment of a well known electroactive

bacteria (G. sulfurreducens) supporting the observed CV shapes for both, turnover and non-

turnover CVs (Harnisch, et al., 2011).

Page 138: PhD Thesis Alessandro Carmona2012

-117-

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

.

The fact that no clear CV shape was found for the rest of the inocula could indicate that in those

electrodes there was no a predominant bacteria in the biofilm but an association of different

microbes part of a mixed culture community.

Page 139: PhD Thesis Alessandro Carmona2012

-118-

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).

8.4 Conclusions

Within this study it is demonstrated the importance of the inoculum and the substrate selection

by analyzing the current production and the formal potentials extracted from cyclic

voltammetry. By this selection, using acetate and lactate-based artificial wastewater as the

bacterial growth medium and real wastewater as inoculum, cyclic voltammograms shapes

similar to pure culture biofilms of G. sulfurreducens were gained (Fricke, et al., 2008). This

raises the question on how distinctive environmental variables (e.g. the bacterial source and the

substrate) influence the bacterial biofilm composition and dynamics. These and further follow-

up questions are under investigation with flow-cytometry, allowing a high-throughput

characterization of natural microbial communities without any previous knowledge on the

bacterial composition (Harnisch, et al., 2011). The monitoring of microbial communities will

use flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring

Page 140: PhD Thesis Alessandro Carmona2012

-119-

dynamics in community structure after the study performed by Günther and co-workers

(Günther, et al., 2011). Additionally, the study will use biostatistics to determine the kind and

strength of the correlation between the presence and abundances of initial and developed

microbial communities. Finally, the bacterial composition of certain subcommunities will be

determined by cell sorting and phylogenetic tools like T-RFLP. Due to the above, the

application of flow-cytometry to electrocatalytic biofilms paves the way to follow the

community dynamics as well as bacterial activity states in response to micro-environmental

changes in high through-put BESs.

Page 141: PhD Thesis Alessandro Carmona2012

-120-

9 Supplementary information: Chapter II

Table S9-1 Comparison of geometric current densities for Shewanella oneidensis Wild-type in

different studies.

jmax-CA/ µA cm-2

Applied E/ V* Ref.

7.9 +0.2 This study

5.1 +0.4 (Babauta, et al., 2011)

2.9 0 (Babauta, et al., 2011)

2.0 +0.2 (Okamoto, et al., 2011)

45.0 +0.2 (Rosenbaum, et al., 2010a)

18.5 +0.043 (Coursolle, et al., 2010)

10.0 -0.195 (Peng, et al., 2010b)

9.7 -0.195 (Peng, et al., 2010a)

17.8 +0.041 (Baron, et al., 2009)

16.0 +0.041 (Marsili, et al., 2008a)

22.9 +0.5 (Cho and Ellington, 2007)

23.6 +0.35 (Cho and Ellington, 2007)

24.3 +0.2 (Cho and Ellington, 2007)

25.7 0 (Cho and Ellington, 2007) *Average maximum current density

Page 142: PhD Thesis Alessandro Carmona2012

-121-

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).

Page 143: PhD Thesis Alessandro Carmona2012

-122-

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.

Page 144: PhD Thesis Alessandro Carmona2012

-123-

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.

Page 145: PhD Thesis Alessandro Carmona2012

-124-

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.

Page 146: PhD Thesis Alessandro Carmona2012

-125-

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.

Page 147: PhD Thesis Alessandro Carmona2012

-126-

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.

Page 148: PhD Thesis Alessandro Carmona2012

-127-

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.

Page 149: PhD Thesis Alessandro Carmona2012

-128-

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)

Page 150: PhD Thesis Alessandro Carmona2012

-129-

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)

Page 151: PhD Thesis Alessandro Carmona2012

-130-

10 Supplementary information: Chapter III

Table S10-1 Comparison of geometric current densities for different strains of Shewanellaceae.

Strain jmax-CA/ µA cm-2

Applied E/ V vs Ag/agCl **Ref.

S. putrefaciens NCTC 10695 2.20 ± 0.62* -0.1 This study

S. putrefaciens NCTC 10695 3.43 ± 0.81* 0.0 This study

S. putrefaciens NCTC 10695 5.31 ± 1.47* +0.1 This study

S. putrefaciens NCTC 10695 7.76 ± 1.44* +0.2 This study

S. putrefaciens NCTC 10695 9.08 ± 1.70* +0.3 This study

S. putrefaciens NCTC 10695 12.03 ± 2.37* +0.4 This study

S. putrefaciens W3-18-1 3.1 MFC at 10 Ω [1]

S. putrefaciens SR-21 0.62 MFC at 1000 Ω [2]

S. putrefaciens ATCC 8071 31.25 MFC at 300 Ω [3]

S. putrefaciens IR-1 0.8 MFC at 1000 Ω [4]

S. putrefaciens IR-1 0.013 MFC at 500 Ω [5]

S. putrefaciens IR-1 0.002 +0.1 [6]

S. oneidensis MR-1 7.9 +0.2 [7]

S. oneidensis MR-1 5.1 +0.4 [8]

S. oneidensis MR-1 2.9 0 [9]

S. oneidensis MR-1 2.0 +0.2 [10]

S. oneidensis MR-1 45.0 +0.2 [11]

S. oneidensis MR-1 1.3 MFC at 10 Ω [12]

S. oneidensis MR-1 18.5 +0.043 [13]

S. oneidensis MR-1 10.0 -0.195 [14]

S. oneidensis MR-1 9.7 -0.195 [15]

S. oneidensis MR-1 17.8 +0.041 [16]

S. oneidensis MR-1 16.0 +0.041 [17]

S. oneidensis MR-1 22.5 +0.041 [18]

S. oneidensis MR-1 22.9 +0.5 [19]

S. oneidensis MR-1 23.6 +0.35 [20]

S. oneidensis MR-1 24.3 +0.2 [21]

S. oneidensis MR-1 25.7 0 [22]

S. oneidensis MR-1 9.6 MFC at 10 Ω [23]

S. oneidensis MR-1 IR-1< j < SR-21 MFC at 1000 Ω [24]

*Average data from chronoamperometric experiments at different applied potentials (vs. Ag/

AgCl) calculated as described in 3.2.4. and its respective standard deviation. **References in

Table: 1: (Bretschger, et al., 2010a); 2: (Kim, et al., 2002); 3: (Park and Zeikus, 2002); 4: (Kim,

et al., 2002); 5: (Kim, et al., 1999d); 6: (Kim, et al., 1999c); 7: (Carmona-Martínez, et al., 2011);

8: (Babauta, et al., 2011); 9: (Babauta, et al., 2011); 10: (Okamoto, et al., 2011); 11:

(Rosenbaum, et al., 2010a); 12: (Bretschger, et al., 2010a); 13: (Coursolle, et al., 2010); 14:

(Peng, et al., 2010b); 15: (Peng, et al., 2010a); 16: (Baron, et al., 2009); 17: (Marsili, et al.,

2008a); 18: (Marsili, et al., 2008a); 19: (Cho and Ellington, 2007); 20: (Cho and Ellington,

2007); 21: (Cho and Ellington, 2007); 22: (Cho and Ellington, 2007); 23: (Gorby, et al., 2006);

24: (Kim, et al., 2002).

Page 152: PhD Thesis Alessandro Carmona2012

-131-

Table S10-1 Comparison of geometric current densities for different strains of Shewanellaceae

(…continuation of Table S10-1).

Strain jmax-CA/ µA cm-2

Applied E/ V vs Ag/agCl *Ref.

S. loihica PV-4 1.5 +0.2 [1]

S. loihica PV-4 4.0 +0.2 [2]

S. loihica PV-4 0.7 MFC at 10 Ω [3]

S. loihica PV-4 100 +0.2 [4]

S. loihica PV-4 1.0 +0.2 [5]

S. loihica PV-4 4.0 +0.2 [6]

S. loihica PV-4 6.0 -0.2 [7]

S. loihica PV-4 0.7 +0.2 [8]

S. loihica PV-4 1.6 +0.201 [9]

S. decolorationis NTOU1 34.0 +0.4 [10]

S. decolorationis NTOU1 97.0 +0.4 [11]

S. decolorationis NTOU1 22 MFC at 800 Ω [12]

S. japonica KMM 3299 22 MFC at 100 kΩ [13]

*References in Table: 1: (Wu, et al., 2011); 2: (Wu, et al., 2011); 3: (Bretschger, et al., 2010a);

4: (Zhao, et al., 2010a); 5: (Zhao, et al., 2010a); 6: (Liu, et al., 2010a); 7: (Liu, et al., 2010a); 8:

(Nakamura, et al., 2009b); 9: (Okamoto, et al., 2009); 10: (Li, et al., 2010); 11: (Li, et al., 2010);

12: (Yang, et al., 2011); 13: (Biffinger, et al., 2011).

Page 153: PhD Thesis Alessandro Carmona2012

-132-

Table S10-2 Shewanella strains used as comparison in Table S10-1 and a description of their

isolation environment.

Strain Environmental characteristics of isolation area *Ref.

S. putrefaciens NCTC 10695 Oil emulsion from a machine shop

[1]

S. putrefaciens ATCC 8071 Responsible for butter putrefaction

[2]

S. putrefaciens W3-18-1 Marine sediment (630 m) in the Pacific Ocean,

Washington Coast, USA

[3]

S. putrefaciens IR-1 Anaerobic habitat in rice paddy field, South Korea

[4]

S. oneidensis MR-1 Anaerobic fresh water sediment in Lake Oneida, NY,

USA

[5]

S. amazonensis SB2B Shallow marine sediment (1 m) in the Amazon River

Delta, Brazil

[6]

S. putrefaciens SR-21 A transposon mutant of MR-1 with loss of Fe(III) and

Mn(IV) reduction

[7]

S. decolorationis NTOU1 Cooling system in an oil refinery in Taiwan

[8]

S. loihica PV-4 Microbial mat located at a hydrothermal vent in South

Rift of Loihi Seamount, Hawaii

[9]

S. oneidensis MR-4 Sea water, oxic zone (5 m) in the Black Sea

[10]

S. japonica KMM 3299 Sea water samples collected from a depth of 0.5-1.5 m

in the Gulf of Peter the Great, Sea of Japan

[11]

Note: Table partially made from information found in (Bretschger, et al., 2010a). *References in

Table: 1: (Pivnick, 1955); 2: (Derby and Hammer, 1931); 3: (Murray, et al., 2001); 4: (Hyun, et

al., 1999); 5: (Myers and Nealson, 1988); 6: (Venkateswaran, et al., 1998); 7: (Beliaev and

Saffarini, 1998); 8: (Chen, et al., 2008); 9: (Gao, et al., 2006); 10: (Nealson, et al., 1991); 11:

(Ivanova, et al., 2001).

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.

Applied E/ V Epc

/ mV Epa

/ mV Ef/ mV ΔE/ mV

-0.1 - - - -

0.0 - - - -

+0.1 - -7 ± 1 - 151 ± 12

+0.2 -129 ± 14 -6 ± 17 -67 ± 15 132 ± 20

+0.3 -126 ± 14 -1 ± 17 -64 ± 15 128 ± 23

+0.4 -118 ± 14 4 ± 16 -57 ± 15 127 ± 22

Epc

: cathodic peak position; Epa

: anodic peak position and Ef: formal potential. Window width

calculated according to Ref. (Firer-Sherwood, et al., 2008a).

Page 154: PhD Thesis Alessandro Carmona2012

-133-

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.

Page 155: PhD Thesis Alessandro Carmona2012

-134-

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

.

Page 156: PhD Thesis Alessandro Carmona2012

-135-

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).

Page 157: PhD Thesis Alessandro Carmona2012

-136-

11 Supplementary information: Chapter VII

11.1 Influence of the buffer capacity

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.

Page 158: PhD Thesis Alessandro Carmona2012

-137-

11.2 Terminal restriction fragment polymorphism (T-RFLP) analysis: Anode biofilm

composition at the different pH values determined by T-RFLP

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).

Page 159: PhD Thesis Alessandro Carmona2012

-138-

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.

Page 160: PhD Thesis Alessandro Carmona2012

-139-

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.

Page 161: PhD Thesis Alessandro Carmona2012

-140-

Conclusion: T-RFLP showed a single peak after RsaI and MspI digestion which was affiliated to

Geobacter sulfurreducens after sequencing for both electrode sets grown at pH 7. The same

phylotype was also found at pH 6 and pH 9 but only in one (high performing) set, whereas in the

respective low performing electrode set no G. sulfurreducens could be detected.

11.3 Terminal restriction fragment polymorphism analysis: Anode chamber community

composition at pH 7 and 9 at different feeding cycles determined by T-RFLP

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.

11.4 Relationship of community composition when cultivated at different pH and under

successive feeding cycles determined by T-RFLP

Page 162: PhD Thesis Alessandro Carmona2012

-141-

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).

Page 163: PhD Thesis Alessandro Carmona2012

-142-

11.5 Flow-cytometric analysis.

11.5.1 Community structure when cultivated at pH 9 at successive feeding cycles

determined by flow cytometry

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).

Page 164: PhD Thesis Alessandro Carmona2012

-143-

11.5.2 Community structure when cultivated at pH 6 at successive feeding cycles

determined by flow cytometry

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).

Page 165: PhD Thesis Alessandro Carmona2012

-144-

11.6 Relationship of community structure when cultivated at different pH and under

successive feeding cycles determined by flow cytometry

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.

Page 166: PhD Thesis Alessandro Carmona2012

-145-

11.7 Statistical Analysis of flow-cytometric data

For identifying community dynamics between feeding cycles and the different pH treatments a

newly developed method named ‘Dalmatian plot analysis’, a combination of image analysis and

multivariate community approach, was used (Bombach, et al., 2011). ‘Dalmatian plots’ are

simplified representations of the usually more complex flow cytometry bivariate plots.

Microbial sub-communities, detected by flow-cytometric measurements, are automatically or

manually encircled by black blots (‘gates’). Subsequently the underlying cytometry plot is

removed from the image. In a next step the resulting black and white Dalmatian plot is

converted into a binary (black-and-white), equal sized, pixel image having gate areas filled in

black and white as background.

For similarity calculation additionally estimates of overlapping areas from all possible binary

combinations are necessary. These are produced by overlaying all image combinations by

simple additive image calculation. For estimating the similarity rate between two images, the

area sums from all gates (the black area of a picture as pixels) from two images and the resulting

overlap of the same combination are estimated. Their similarity is then estimated using a

modified Jaccard index given by:

21

21

21

))2()1((1

AA

AA

AAOverlap

OverlapAAS

with similarity SA1A2 between two images A1 and A2, the sum of all gates in pixels counts of

picture A1 and A2, respectively and OverlapA1A2 the pixel sums of the overlap of image A1 and

A2. In this approach only presence and absence of sub-communities are regarded irrespective of

abundances within gates but thus enabling equal priority of all emerging sub-communities

therein.

Overlay creation and gate area calculation were automatically done with ImageJ Version 1.43

(http://rsb.info.nih.gov/ij). The similarity results of all possible combinations were then

transferred into a triangular similarity matrix. The final n-MDS analysis and a cluster analysis

based on the similarity matrix were accomplished under R Version 2.12.1 (Development-Core-

Team, 2010). For estimating overlays and estimating pixel areas of the single Dalmatian plots

and their overlays automatically a script for ImageJ was written.

Page 167: PhD Thesis Alessandro Carmona2012

-146-

For estimating similarities and conduction of n-MDS and a cluster analysis directly from an

output of pixel counts created by ImageJ additionally a script for R was developed. Both scripts

are freely available and can be purchased by contacting the authors.

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).

11.8 Biofilm detachment

Figure S11-11 Photograph of the detachment of a pH 7 grown biofilm from an electrode due to

extreme pH-conditions (pH 11).

Page 168: PhD Thesis Alessandro Carmona2012

-147-

11.9 Multivariate statistical analysis of the flow-cytometric pattern using n-MDS-plots

The complex community and biofilm dynamics in response to micro-environmental changes like

pH-value and cycle conditions can be analyzed using Dalmatian plot based n-MDS analysis (for

method details see (Bombach, et al., 2011) and the following information presented in this

Chapter). In brief, first the microbial community changes detected via flow-cytometry are

visualized using Dalmatian-Plots. These plots are simplified representations of the more

complex density plots (see Figure 7-4), i.e. the raw-data, in which every dot represents a signal

event in the cytometric measurement. Within the derived Dalmatian plots black areas represent

identified microbial sub-communities (see below for an example and further explanation). Thus,

based on the number and position of these areas, representing sub-communities, every

Dalmatian plot can be considered as “fingerprint” of the bacterial culture for a certain point of

time and condition. Subsequently, based on these Dalmatian fingerprints, a similarity alignment

of the flow-cytometric data can be performed, which results in the n-MDS plot (Figure 7-5).

Roughly, within this plot, which is commonly used for the analyses of complex data sets, similar

samples are grouped together whereas dissimilar ones are grouped more distant. The stress-

value thereby provides a control measure for assessing the chance of data-misinterpretation. The

obtained stress-value for our analyses of 17.75 thereby clearly shows that the grouping of the

samples leads to no misinterpretation (see Figure 7-5).

Page 169: PhD Thesis Alessandro Carmona2012

-148-

12 References

Abdo, Z., Schüette, U.M.E., Bent, S.J., Williams, C.J., Forney, L.J., Joyce, P., Statistical methods for

characterizing diversity of microbial communities by analysis of terminal restriction fragment

length polymorphisms of 16S rRNA genes, Environmental Microbiology, 2006, 8 (5): 929-938.

Adachi, M., Shimomura, T., Komatsu, M., Yakuwa, H., Miya, A., A novel mediator-polymer-modified

anode for microbial fuel cells, Chemical Communications, 2008, (17): 2055-2057.

Aelterman, P., Rabaey, K., Clauwaert, P., Verstraete, W., Microbial fuel cells for wastewater treatment,

in: Water Science and Technology, 2006, pp. 9-15.

Agarwal, S., Wendorff, J.H., Greiner, A., Use of electrospinning technique for biomedical applications,

Polymer, 2008, 49 (26): 5603-5621.

Angenent, L.T., Karim, K., Al-Dahhan, M.H., Wrenn, B.A., Domínguez-Espinosa, R., Production of

bioenergy and biochemicals from industrial and agricultural wastewater, Trends in

Biotechnology, 2004, 22 (9): 477-485.

Atlas, R.M., Handbook of Microbiological Media, 3th ed., CRC Press LLC, Florida, 1993.

Aulenta, F., Catervi, A., Majone, M., Panero, S., Reale, P., Rossetti, S., Electron transfer from a solid-

state electrode assisted by methyl viologen sustains efficient microbial reductive dechlorination

of TCE, Environmental Science & Technology, 2007, 41 (7): 2554-2559.

Babauta, J.T., Nguyen, H.D., Beyenal, H., Redox and pH Microenvironments within Shewanella

oneidensis MR-1 Biofilms Reveal an Electron Transfer Mechanism, Environmental Science &

Technology, 2011, 45 (15): 6654-6660.

Balch, W.E., Fox, G.E., Magrum, L.J., Methanogens: reevaluation of a unique biological group,

Microbiological Reviews, 1979, 43 (2): 260-296.

Bansal, D., Meyer, B., Salomon, M., Gelled membranes for Li and Li-ion batteries prepared by

electrospinning, Journal of Power Sources, 2008, 178 (2): 848-851.

Bard, A.J., Inzelt, G., Scholz, F., Electrochemical Dictionary, Springer London, Limited, 2008.

Baron, D., LaBelle, E., Coursolle, D., Gralnick, J.A., Bond, D.R., Electrochemical Measurement of

Electron Transfer Kinetics by Shewanella oneidensis MR-1, Journal of Biological Chemistry,

2009, 284 (42): 28865-28873.

Beliaev, A.S., Klingeman, D.M., Klappenbach, J.A., Wu, L., Romine, M.F., Tiedje, J.M., Nealson, K.H.,

Fredrickson, J.K., Zhou, J., Global Transcriptome Analysis of Shewanella oneidensis MR-1

Exposed to Different Terminal Electron Acceptors, J. Bacteriol., 2005, 187 (20): 7138-7145.

Beliaev, A.S., Saffarini, D.A., Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein

Required for Fe(III) and Mn(IV) Reduction, Journal of Bacteriology, 1998, 180 (23): 6292-

6297.

Bennetto, H.P., Stirling, J.L., Tanaka, K., Vega, C.A., Anodic reactions in microbial fuel cells,

Biotechnology and Bioengineering, 1983, 25 (2): 559-568.

Bhatnagar, D., Xu, S., Fischer, C., Arechederra, R.L., Minteer, S.D., Mitochondrial biofuel cells:

expanding fuel diversity to amino acids, Physical Chemistry Chemical Physics, 2011, 13 (1): 86-

92.

Biffinger, J.C., Fitzgerald, L.A., Ray, R., Little, B.J., Lizewski, S.E., Petersen, E.R., Ringeisen, B.R.,

Sanders, W.C., Sheehan, P.E., Pietron, J.J., Baldwin, J.W., Nadeau, L.J., Johnson, G.R., Ribbens,

M., Finkel, S.E., Nealson, K.H., The utility of Shewanella japonica for microbial fuel cells,

Bioresource Technology, 2010, 102 (1): 290-297.

Biffinger, J.C., Fitzgerald, L.A., Ray, R., Little, B.J., Lizewski, S.E., Petersen, E.R., Ringeisen, B.R.,

Sanders, W.C., Sheehan, P.E., Pietron, J.J., Baldwin, J.W., Nadeau, L.J., Johnson, G.R., Ribbens,

M., Finkel, S.E., Nealson, K.H., The utility of Shewanella japonica for microbial fuel cells,

Bioresource Technology, 2011, 102 (1): 290-297.

Biffinger, J.C., Pietron, J., Bretschger, O., Nadeau, L.J., Johnson, G.R., Williams, C.C., Nealson, K.H.,

Ringeisen, B.R., The influence of acidity on microbial fuel cells containing Shewanella

oneidensis, Biosensors and Bioelectronics, 2008, 24 (4): 900-905.

Biffinger, J.C., Ray, R., Little, B.J., Fitzgerald, L.A., Ribbens, M., Finkel, S.E., Ringeisen, B.R.,

Simultaneous analysis of physiological and electrical output changes in an operating microbial

Page 170: PhD Thesis Alessandro Carmona2012

-149-

fuel cell with Shewanella oneidensis, Biotechnology and Bioengineering, 2009, 103 (3): 524-

531.

Bombach, P., Hübschmann, T., Fetzer, I., Kleinsteuber, S., Geyer, R., Harms, H., Müller, S., Resolution

of natural microbial community dynamics by community fingerprinting, flow cytometry, and

trend interpretation analysis, in: Advances in Biochemical Engineering/Biotechnology, 2011,

pp. 151-181.

Bond, D.R., Holmes, D.E., Tender, L.M., Lovley, D.R., Electrode-Reducing Microorganisms That

Harvest Energy from Marine Sediments, Science, 2002, 295 (5554): 483-485.

Borole, A.P., O'Neill, H., Tsouris, C., Cesar, S., A microbial fuel cell operating at low pH using the

acidophile Acidiphilium cryptum, Biotechnology Letters, 2008, 30 (8): 1367-1372.

Bouhenni, R.A., Vora, G.J., Biffinger, J.C., Shirodkar, S., Brockman, K., Ray, R., Wu, P., Johnson, B.J.,

Biddle, E.M., Marshall, M.J., Fitzgerald, L.A., Little, B.J., Fredrickson, J.K., Beliaev, A.S.,

Ringeisen, B.R., Saffarini, D.A., The role of Shewanella oneidensis MR-1 outer surface

structures in extracellular electron transfer, Electroanalysis, 2010, 22 (7-8): 856-864.

Bretschger, O., Cheung, A.C.M., Mansfeld, F., Nealson, K.H., Comparative Microbial Fuel Cell

Evaluations of Shewanella spp, Electroanalysis, 2010a, 22 (7-8): 883-894.

Bretschger, O., Gorby, Y.A., Nealson, K.H., A Survey Of Direct Electron Transfer from Microbes to

Electronically Active Surfaces, in: K. Rabaey, L. Angenent, U. Schroder, J. Keller (Eds.)

Bioelectrochemical Systems: from Extracellular Electron Transfer to Biotechnological

Application, 2010b.

Bretschger, O., Obraztsova, A., Sturm, C.A., In, S.C., Gorby, Y.A., Reed, S.B., Culley, D.E., Reardon,

C.L., Barua, S., Romine, M.F., Zhou, J., Beliaev, A.S., Bouhenni, R., Saffarini, D., Mansfeld, F.,

Kim, B.H., Fredrickson, J.K., Nealson, K.H., Current production and metal oxide reduction by

Shewanella oneidensis MR-1 wild type and mutants, Applied and Environmental Microbiology,

2007, 73 (21): 7003-7012.

Busalmen, J.P., Esteve-Núñez, A., Berná, A., Feliu, J.M., C-type cytochromes wire electricity-producing

bacteria to electrodes, Angewandte Chemie - International Edition, 2008, 47 (26): 4874-4877.

Caccavo, F., Jr, Lonergan, D.J., Lovley, D.R., Davis, M., Stolz, J.F., McInerney, M.J., Geobacter

sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing

microorganism, Appl. Environ. Microbiol., 1994, 60 (10): 3752-3759.

Caccavo Jr, F., Coates, J.D., Rossello-Mora, R.A., Ludwig, W., Schleifer, K.H., Lovley, D.R.,

McInerney, M.J., Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe (III)-

reducing bacterium, Archives of Microbiology, 1996, 165 (6): 370-376.

Cao, X., Huang, X., Liang, P., Xiao, K., Zhou, Y., Zhang, X., Logan, B.E., A New Method for Water

Desalination Using Microbial Desalination Cells, Environmental Science & Technology, 2009,

43 (18): 7148-7152.

Cao, Y., Hu, Y., Sun, J., Hou, B., Explore various co-substrates for simultaneous electricity generation

and Congo red degradation in air-cathode single-chamber microbial fuel cell,

Bioelectrochemistry, 2010, 79 (1): 71-76.

Carmona-Martínez, A.A., Harnisch, F., Fitzgerald, L.A., Biffinger, J.C., Ringeisen, B.R., Schröder, U.,

Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis MR-1 and

nanofilament and cytochrome knock-out mutants, Bioelectrochemistry, 2011, 81 (2): 74-80.

Catal, T., Li, K., Bermek, H., Liu, H., Electricity production from twelve monosaccharides using

microbial fuel cells, Journal of Power Sources, 2008a, 175 (1): 196-200.

Catal, T., Xu, S., Li, K., Bermek, H., Liu, H., Electricity generation from polyalcohols in single-chamber

microbial fuel cells, Biosensors and Bioelectronics, 2008b, 24 (4): 849-854.

Cervantes, F.J., Vu-Thi-Thu, L., Lettinga, G., Field, J.A., Quinone-respiration improves dechlorination

of carbon tetrachloride by anaerobic sludge, Applied Microbiology and Biotechnology, 2004, 64

(5): 702-711.

Chae, K.-J., Choi, M.-J., Lee, J.-W., Kim, K.-Y., Kim, I.S., Effect of different substrates on the

performance, bacterial diversity, and bacterial viability in microbial fuel cells, Bioresource

Technology, 2009, 100 (14): 3518-3525.

Chang, I.S., Moon, H., Bretschger, O., Jang, J.K., Park, H.I., Nealson, K.H., Kim, B.H.,

Electrochemically active bacteria (EAB) and mediator-less microbial fuel cells, Journal of

Microbiology and Biotechnology, 2006, 16 (2): 163-177.

Page 171: PhD Thesis Alessandro Carmona2012

-150-

Chang, I.S., Moon, H., Jang, J.K., Kim, B.H., Improvement of a microbial fuel cell performance as a

BOD sensor using respiratory inhibitors, Biosensors and Bioelectronics, 2005, 20 (9): 1856-

1859.

Chaudhuri, S.K., Lovley, D.R., Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells, Nat Biotech, 2003, 21 (10): 1229-1232.

Chen, C.-H., Chang, C.-F., Ho, C.-H., Tsai, T.-L., Liu, S.-M., Biodegradation of crystal violet by a

Shewanella sp. NTOU1, Chemosphere, 2008, 72 (11): 1712-1720.

Chen, S., Hou, H., Harnisch, F., Patil, S.A., Carmona-Martinez, A.A., Agarwal, S., Zhang, Y., Sinha-

Ray, S., Yarin, A.L., Greiner, A., Schroder, U., Electrospun and solution blown three-

dimensional carbon fiber nonwovens for application as electrodes in microbial fuel cells, Energy

& Environmental Science, 2011, 4 (4): 1417-1421.

Cheng, S., Liu, H., Logan, B.E., Increased performance of single-chamber microbial fuel cells using an

improved cathode structure, Electrochemistry Communications, 2006, 8 (3): 489-494.

Cheng, S., Logan, B.E., Ammonia treatment of carbon cloth anodes to enhance power generation of

microbial fuel cells, Electrochemistry Communications, 2007, 9 (3): 492-496.

Cho, E.J., Ellington, A.D., Optimization of the biological component of a bioelectrochemical cell,

Bioelectrochemistry, 2007, 70 (1): 165-172.

Coates, J.D., Ellis, D.J., Gaw, C.V., Lovley, D.R., Geothrix fermentans gen. nov., sp. nov., a novel

Fe(III)-reducing bacterium from a hydrocarbon-contaminated aquifer, International Journal of

Systematic and Evolutionary Microbiology, 1999, 49 (4): 1615-1622.

Cohen, B., The Bacterial Culture as an Electrical Half-Cell, Journal of Bacteriology, 1931, 21 (1): 1-60.

Cooney, M.J., Svoboda, V., Lau, C., Martin, G., Minteer, S.D., Enzyme catalysed biofuel cells, Energy

& Environmental Science, 2008, 1 (3): 320-337.

Cornell, R.M., Schwertmann, U., The Iron Oxides: Structure, Properties, Reactions, Occurrences and

Uses, John Wiley & Sons, 2007.

Cournet, A., Délia, M.-L., Bergel, A., Roques, C., Bergé, M., Electrochemical reduction of oxygen

catalyzed by a wide range of bacteria including Gram-positive, Electrochemistry

Communications, 2010, 12 (4): 505-508.

Coursolle, D., Baron, D.B., Bond, D.R., Gralnick, J.A., The Mtr Respiratory Pathway is Essential for

Reducing Flavins and Electrodes in Shewanella oneidensis, J. Bacteriol., 2010: JB.00925-00909.

Delaney, G.M., Bennetto, H.P., Mason, J.R., Roller, S.D., Stirling, J.L., Thurston, C.F., Electron-transfer

coupling in microbial fuel cells. 2. performance of fuel cells containing selected

microorganism—mediator—substrate combinations, Journal of Chemical Technology and

Biotechnology. Biotechnology, 1984, 34 (1): 13-27.

Derby, H.A., Hammer, B.W., Bacteriology of butter: Bacteriological studies on surface taint butter,

Agricultural Experiment Station, Iowa State College of Agriculture and Mechanic Arts, 1931.

Development-Core-Team, A language and environment for statistical computing, in, Foundation for

Statistical Computing, Vienna, Austria, 2010.

Ditzig, J., Liu, H., Logan, B.E., Production of hydrogen from domestic wastewater using a

bioelectrochemically assisted microbial reactor (BEAMR), International Journal of Hydrogen

Energy, 2007, 32 (13): 2296-2304.

Doong, R.A., Schink, B., Cysteine-mediated reductive dissolution of poorly crystalline iron (III) oxides

by Geobacter sulfurreducens, Environmental Science & Technology, 2002, 36 (13): 2939-2945.

Ducommun, R., Favre, M.-F., Carrard, D., Fischer, F., Outward electron transfer by Saccharomyces

cerevisiae monitored with a bi-cathodic microbial fuel cell-type activity sensor, Yeast, 2010, 27

(3): 139-148.

Eggleston, C.M., Vörös, J., Shi, L., Lower, B.H., Droubay, T.C., Colberg, P.J.S., Binding and direct

electrochemistry of OmcA, an outer-membrane cytochrome from an iron reducing bacterium,

with oxide electrodes : A candidate biofuel cell system, Anglais, 2008, 361 (3): 769-777.

El-Naggar, M.Y., Wanger, G., Leung, K.M., Yuzvinsky, T.D., Southam, G., Yang, J., Lau, W.M.,

Nealson, K.H., Gorby, Y.A., Electrical transport along bacterial nanowires from Shewanella

oneidensis MR-1, Proceedings of the National Academy of Sciences, 2010, 107 (42): 18127-

18131.

Fan, Y., Sharbrough, E., Liu, H., Quantification of the Internal Resistance Distribution of Microbial Fuel

Cells, Environmental Science & Technology, 2008, 42 (21): 8101-8107.

Page 172: PhD Thesis Alessandro Carmona2012

-151-

Fenchel, T., Blackburn, T.H., Bacteria and mineral cycling, Academic Press, 1979.

Firer-Sherwood, M., Pulcu, G., Elliott, S., Electrochemical interrogations of the Mtr cytochromes from

Shewanella: opening a potential window, Journal of Biological Inorganic Chemistry, 2008a, 13

(6): 849-854.

Firer-Sherwood, M., Pulcu, G.S., Elliott, S.J., Electrochemical interrogations of the Mtr cytochromes

from Shewanella: Opening a potential window, Journal of Biological Inorganic Chemistry,

2008b, 13 (6): 849-854.

Flemming, H.C., Wingender, J., The biofilm matrix, Nature Reviews Microbiology, 2010, 8 (9): 623-633.

Fourmond, V., Hoke, K., Heering, H.A., Baffert, C., Leroux, F., Bertrand, P., Léger, C., SOAS: A free

program to analyze electrochemical data and other one-dimensional signals, Bioelectrochemistry,

2009, 76 (1-2): 141-147.

Franks, A.E., Malvankar, N., Nevin, K.P., Bacterial biofilms: the powerhouse of a microbial fuel cell,

Biofuels, 2010, 1 (4): 589-604.

Franks, A.E., Nevin, K.P., Jia, H., Izallalen, M., Woodard, T.L., Lovley, D.R., Novel strategy for three-

dimensional real-time imaging of microbial fuel cell communities: Monitoring the inhibitory

effects of proton accumulation within the anode biofilm, Energy and Environmental Science,

2009, 2 (1): 113-119.

Fredrickson, J.K., Gorby, Y.A., Environmental processes mediated by iron-reducing bacteria, Current

Opinion in Biotechnology, 1996, 7 (3): 287-294.

Fredrickson, J.K., Kostandarithes, H.M., Li, S.W., Plymale, A.E., Daly, M.J., Reduction of Fe (III), Cr

(VI), U (VI), and Tc (VII) by deinococcus radiodurans R1, Applied and Environmental

Microbiology, 2000a, 66 (5): 2006.

Fredrickson, J.K., Romine, M.F., Beliaev, A.S., Auchtung, J.M., Driscoll, M.E., Gardner, T.S., Nealson,

K.H., Osterman, A.L., Pinchuk, G., Reed, J.L., Rodionov, D.A., Rodrigues, J.L.M., Saffarini,

D.A., Serres, M.H., Spormann, A.M., Zhulin, I.B., Tiedje, J.M., Towards environmental systems

biology of Shewanella, Nat Rev Micro, 2008, 6 (8): 592-603.

Fredrickson, J.K., Zachara, J.M., Kennedy, D.W., Duff, M.C., Gorby, Y.A., Li, S.W., Krupka, K.M.,

Reduction of U (VI) in goethite ([alpha]-FeOOH) suspensions by a dissimilatory metal-reducing

bacterium, Geochimica et Cosmochimica Acta, 2000b, 64 (18): 3085-3098.

Fricke, K., Harnisch, F., Schroder, U., On the use of cyclic voltammetry for the study of anodic electron

transfer in microbial fuel cells, Energy & Environmental Science, 2008, 1 (1): 144-147.

Friedheim, E., Michaelis, L., Potentiometric study of pyocyanine, Journal of Biological Chemistry, 1931,

91 (1): 355-368.

Fu, L., You, S.-J., Zhang, G.-q., Yang, F.-L., Fang, X.-h., Degradation of azo dyes using in-situ Fenton

reaction incorporated into H2O2-producing microbial fuel cell, Chemical Engineering Journal,

2010, 160 (1): 164-169.

Gandhi, M., Srikar, R., Yarin, A.L., Megaridis, C.M., Gemeinhart, R.A., Mechanistic examination of

protein release from polymer nanofibers, Molecular Pharmaceutics, 2009, 6 (2): 641-647.

Gao, H., Obraztova, A., Stewart, N., Popa, R., Fredrickson, J.K., Tiedje, J.M., Nealson, K.H., Zhou, J.,

Shewanella loihica sp. nov., isolated from iron-rich microbial mats in the Pacific Ocean,

International Journal of Systematic and Evolutionary Microbiology, 2006, 56 (8): 1911-1916.

Gil, G.-C., Chang, I.-S., Kim, B.H., Kim, M., Jang, J.-K., Park, H.S., Kim, H.J., Operational parameters

affecting the performannce of a mediator-less microbial fuel cell, Biosensors and Bioelectronics,

2003, 18 (4): 327-334.

Giraffa, G., Rossetti, L., Neviani, E., An evaluation of chelex-based DNA purification protocols for the

typing of lactic acid bacteria, Journal of Microbiological Methods, 2000, 42 (2): 175-184.

Gorby, Y.A., Yanina, S., McLean, J.S., Rosso, K.M., Moyles, D., Dohnalkova, A., Beveridge, T.J.,

Chang, I.S., Kim, B.H., Kim, K.S., Culley, D.E., Reed, S.B., Romine, M.F., Saffarini, D.A., Hill,

E.A., Shi, L., Elias, D.A., Kennedy, D.W., Pinchuk, G., Watanabe, K., Ishii, S.i., Logan, B.,

Nealson, K.H., Fredrickson, J.K., Electrically conductive bacterial nanowires produced by

Shewanella oneidensis strain MR-1 and other microorganisms, Proceedings of the National

Academy of Sciences, 2006, 103 (30): 11358-11363.

Gralnick, J.A., Newman, D.K., Extracellular respiration, Molecular Microbiology, 2007, 65 (1): 1-11.

Greiner, A., Wendorff, J.H., Electrospinning: A fascinating method for the preparation of ultrathin fibers,

Angewandte Chemie - International Edition, 2007, 46 (30): 5670-5703.

Page 173: PhD Thesis Alessandro Carmona2012

-152-

Günther, S., Koch, C., Hübschmann, T., Röske, I., Müller, R.A., Bley, T., Harms, H., Müller, S.,

Correlation of community dynamics and process parameters as a tool for the prediction of the

stability of wastewater treatment, 2011.

Guo, Q., Zhou, X., Li, X., Chen, S., Seema, A., Greiner, A., Hou, H., Supercapacitors based on hybrid

carbon nanofibers containing multiwalled carbon nanotubes, Journal of Materials Chemistry,

2009, 19 (18): 2810-2816.

Harnisch, F., Koch, C., Patil, S.A., Hübschmann, T., Müller, S., Schröder, U., Revealing the

electrochemically driven selection in natural community derived microbial biofilms using flow-

cytometry, Energy and Environmental Science, 2011, 4 (4): 1265-1267.

Harnisch, F., Rabaey, K., The diversity of techniques to study electrochemically active biofilms

highlights the need for standardization, ChemSusChem, 2012, special issue "Microbial fuel cell":

p. submitted.

Harnisch, F., Schröder, U., Selectivity versus mobility: Separation of anode and cathode in microbial

bioelectrochemical systems, ChemSusChem, 2009, 2 (10): 921-926.

Harnisch, F., Schröder, U., From MFC to MXC: Chemical and biological cathodes and their potential for

microbial bioelectrochemical systems, Chemical Society Reviews, 2010, 39 (11): 4433-4448.

Harris, H.W., El-Naggar, M.Y., Bretschger, O., Ward, M.J., Romine, M.F., Obraztsova, A.Y., Nealson,

K.H., Electrokinesis is a microbial behavior that requires extracellular electron transport,

Proceedings of the National Academy of Sciences, 2010, 107 (1): 326-331.

Hartshorne, R.S., Jepson, B.N., Clarke, T.A., Field, S.J., Fredrickson, J., Zachara, J.M., Shi, L., Butt,

J.N., Richardson, D.J., Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme

cytochrome involved in respiratory electron transport to extracellular electron acceptors, J. Biol.

Inorg. Chem., 2007, 12: 1083-1094.

Hartshorne, R.S., Reardon, C.L., Ross, D., Nuester, J., Clarke, T.A., Gates, A.J., Mills, P.C., Fredrickson,

J.K., Zachara, J.M., Shi, L., Beliaev, A.S., Marshall, M.J., Tien, M., Brantley, S., Butt, J.N.,

Richardson, D.J., Characterization of an electron conduit between bacteria and the extracellular

environment, Proceedings of the National Academy of Sciences, 2009, 106 (52): 22169-22174.

Hashsham, S.A., Freedman, D.L., Enhanced biotransformation of carbon tetrachloride by

Acetobacterium woodii upon addition of hydroxocobalamin and fructose, Applied and

Environmental Microbiology, 1999, 65 (10): 4537-4542.

He, G., Gu, Y., He, S., Schröder, U., Chen, S., Hou, H., Effect of fiber diameter on the behavior of

biofilm and anodic performance of fiber electrodes in microbial fuel cells, Bioresource

Technology, 2011, 102 (22): 10763-10766.

He, Z., Huang, Y., Manohar, A.K., Mansfeld, F., Effect of electrolyte pH on the rate of the anodic and

cathodic reactions in an air-cathode microbial fuel cell, Bioelectrochemistry, 2008, 74 (1): 78-82.

He, Z., Minteer, S.D., Angenent, L.T., Electricity Generation from Artificial Wastewater Using an

Upflow Microbial Fuel Cell, Environmental Science & Technology, 2005, 39 (14): 5262-5267.

Heidelberg, J.F., Paulsen, I.T., Nelson, K.E., Gaidos, E.J., Nelson, W.C., Read, T.D., Eisen, J.A.,

Seshadri, R., Ward, N., Methe, B., Clayton, R.A., Meyer, T., Tsapin, A., Scott, J., Beanan, M.,

Brinkac, L., Daugherty, S., DeBoy, R.T., Dodson, R.J., Durkin, A.S., Haft, D.H., Kolonay, J.F.,

Madupu, R., Peterson, J.D., Umayam, L.A., White, O., Wolf, A.M., Vamathevan, J., Weidman,

J., Impraim, M., Lee, K., Berry, K., Lee, C., Mueller, J., Khouri, H., Gill, J., Utterback, T.R.,

McDonald, L.A., Feldblyum, T.V., Smith, H.O., Craig Venter, J., Nealson, K.H., Fraser, C.M.,

Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis,

Nature Biotechnology, 2002, 20 (11): 1118-1123.

Hellmann, C., Greiner, A., Wendorff, J.H., Design of pheromone releasing nanofibers for plant

protection, Polymers for Advanced Technologies, 2011, 22 (4): 407-413.

Hernandez, M.E., Newman, D.K., Extracellular electron transfer, Cellular and Molecular Life Sciences,

2001, 58 (11): 1562-1571.

Holmes, D.E., Bond, D.R., Lovley, D.R., Electron Transfer by Desulfobulbus propionicus to Fe(III) and

Graphite Electrodes, Appl. Environ. Microbiol., 2004a, 70 (2): 1234-1237.

Holmes, D.E., Nicoll, J.S., Bond, D.R., Lovley, D.R., Potential Role of a Novel Psychrotolerant Member

of the Family Geobacteraceae, Geopsychrobacter electrodiphilus gen. nov., sp. nov., in

Electricity Production by a Marine Sediment Fuel Cell, Applied and Environmental

Microbiology, 2004b, 70 (10): 6023-6030.

Page 174: PhD Thesis Alessandro Carmona2012

-153-

Hong, Yi, G., Jun, G.U.O., Xu, Zhi, C., Mei, Y., Sun, Guo, P., Humic substances act as electron acceptor

and redox mediator for microbial dissimilatory azoreduction by Shewanella decolorationis S12,

Journal of Microbiology and Biotechnology, 2007, 17: 10.

Hong, S.W., Chang, I.S., Choi, Y.S., Chung, T.H., Experimental evaluation of influential factors for

electricity harvesting from sediment using microbial fuel cell, Bioresource Technology, 2009,

100 (12): 3029-3035.

Hou, H., Reneker, D.H., Carbon Nanotubes on Carbon Nanofibers: A Novel Structure Based on

Electrospun Polymer Nanofibers, Advanced Materials, 2004, 16 (1): 69-73.

Huang, J., Sun, B., Zhang, X., Electricity generation at high ionic strength in microbial fuel cell by a

newly isolated Shewanella marisflavi EP1, Applied Microbiology and Biotechnology, 2010, 85

(4): 1141-1149.

Hyun, M.S., Kim, B.H., Chang, I.S., Park, H.S., Kim, H.J., Kim, G.T., Kim, M.A., Park, D.H., Isolation

and Identification of an Anaerobic Dissimilatory Fe(III)-Reducing Bacterium, Shewanella

putrefaciens IR-1, Journal of Microbiology, 1999, 37 (4): 206-212.

Ieropoulos, I., Winfield, J., Greenman, J., Effects of flow-rate, inoculum and time on the internal

resistance of microbial fuel cells, Bioresource Technology, 2010, 101 (10): 3520-3525.

Inoue, K., Leang, C., Franks, A.E., Woodard, T.L., Nevin, K.P., Lovley, D.R., Specific localization of

the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter

sulfurreducens, Environmental Microbiology Reports, 2011, 3 (2): 211-217.

Ivanova, E.P., Sawabe, T., Gorshkova, N.M., Svetashev, V.I., Mikhailov, V.V., Nicolau, D.V., Christen,

R., Shewanella japonica sp. nov, International Journal of Systematic and Evolutionary

Microbiology, 2001, 51 (3): 1027-1033.

Jadhav, G.S., Ghangrekar, M.M., Performance of microbial fuel cell subjected to variation in pH,

temperature, external load and substrate concentration, Bioresource Technology, 2009, 100 (2):

717-723.

Jensen, H.M., Albers, A.E., Malley, K.R., Londer, Y.Y., Cohen, B.E., Helms, B.A., Weigele, P., Groves,

J.T., Ajo-Franklin, C.M., Engineering of a synthetic electron conduit in living cells, Proceedings

of the National Academy of Sciences, 2010, 107 (45): 19213-19218.

Ji, L., Zhang, X., Fabrication of porous carbon nanofibers and their application as anode materials for

rechargeable lithium-ion batteries, Nanotechnology, 2009, 20 (15).

Jiang, D., Li, B., Jia, W., Lei, Y., Effect of Inoculum Types on Bacterial Adhesion and Power Production

in Microbial Fuel Cells, Applied Biochemistry and Biotechnology, 160 (1): 182-196.

Jiang, X., Hu, J., Fitzgerald, L.A., Biffinger, J.C., Xie, P., Ringeisen, B.R., Lieber, C.M., Probing

electron transfer mechanisms in Shewanella oneidensis MR-1 using a nanoelectrode platform

and single-cell imaging, Proceedings of the National Academy of Sciences, 2010, 107 (39):

16806-16810.

Jiao, Y., Qian, F., Li, Y., Wang, G., Saltikov, C.W., Gralnick, J.A., Deciphering the Electron Transport

Pathway for Graphene Oxide Reduction by Shewanella oneidensis MR-1, Journal of

Bacteriology, 2011, 193 (14): 3662-3665.

Jung, S., Regan, J., Comparison of anode bacterial communities and performance in microbial fuel cells

with different electron donors, Applied Microbiology and Biotechnology, 2007, 77 (2): 393-402.

Kaden, J., S. Galushko, A., Schink, B., Cysteine-mediated electron transfer in syntrophic acetate

oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes, Archives of

Microbiology, 2002, 178 (1): 53-58.

Katuri, K., Ferrer, M.L., Gutierrez, M.C., Jimenez, R., del Monte, F., Leech, D., Three-dimensional

microchanelled electrodes in flow-through configuration for bioanode formation and current

generation, Energy & Environmental Science, 2011, 4 (10): 4201-4210.

Katuri, K.P., Kavanagh, P., Rengaraj, S., Leech, D., Geobacter sulfurreducens biofilms developed under

different growth conditions on glassy carbon electrodes: Insights using cyclic voltammetry,

Chemical Communications, 2010, 46 (26): 4758-4760.

Keck, A., Rau, J., Reemtsma, T., Mattes, R., Stolz, A., Klein, J., Identification of quinoide redox

mediators that are formed during the degradation of naphthalene-2-sulfonate by Sphingomonas

xenophaga BN6, Applied and Environmental Microbiology, 2002, 68 (9): 4341-4349.

Keller, J., Rozendal, R.A., Angenent, L., Schröder, U., Lens, P., Rabaey, K., Outlook: Research

Directions and New Applications for Bes, in: K. Rabaey, L. Angenent, U. Schroder, J. Keller

Page 175: PhD Thesis Alessandro Carmona2012

-154-

(Eds.) Bioelectrochemical Systems: from Extracellular Electron Transfer to Biotechnological

Application, 2010, pp. 449-465.

Khoa Ly, H., Sezer, M., Wisitruangsakul, N., Feng, J.-J., Kranich, A., Millo, D., Weidinger, I.M.,

Zebger, I., Murgida, D.H., Hildebrandt, P., Surface-enhanced vibrational spectroscopy for

probing transient interactions of proteins with biomimetic interfaces: electric field effects on

structure, dynamics and function of cytochrome c, FEBS Journal, 2011, 278 (9): 1382-1390.

Kim, B., Chang, I., Hyun, M., Kim, H., Park, H., A biofuel cell using wastewater and active sludge for

wastewater treatment, A Biofuel Cell Using Wastewater and Active Sludge for Wastewater

Treatment, 2001, EP1232123 / WO WO0104061 (EP20000911467 20000317).

Kim, B.H., Ikeda, T., Park, H.S., Kim, H.J., Hyun, M.S., Kano, K., Takagi, K., Tatsumi, H.,

Electrochemical activity of an Fe(III)-reducing bacterium, Shewanella putrefaciens IR-1, in the

presence of alternative electron acceptors, Biotechnology Techniques, 1999a, 13 (7): 475-478.

Kim, B.H., Kim, H.J., Hyun, M.S., Park, D.H., Direct electrode reaction of Fe(III)-reducing bacterium,

Shewanella putrefaciens, Journal of Microbiology and Biotechnology, 1999b, 9 (2): 127-131.

Kim, B.H., Kim, H.J., Hyun, M.S., Park, H.S., Direct electrode reaction of Fe(III)-Reducing Bacterium ,

Shewanella putrefaciens, Journal of Microbiology and Biotechnology, 1999c, 9 (2): 127-131.

Kim, B.H., Park, H.S., Kim, H.J., Kim, G.T., Chang, I.S., Lee, J., Phung, N.T., Enrichment of microbial

community generating electricity using a fuel-cell-type electrochemical cell, Applied

Microbiology and Biotechnology, 2004, 63 (6): 672-681.

Kim, C., Ngoc, B.T.N., Yang, K.S., Kojima, M., Kim, Y.A., Kim, Y.J., Endo, M., Yang, S.C., Self-

Sustained Thin Webs Consisting of Porous Carbon Nanofibers for Supercapacitors via the

Electrospinning of Polyacrylonitrile Solutions Containing Zinc Chloride, Advanced Materials,

2007, 19 (17): 2341-2346.

Kim, C., Yang, K.S., Electrochemical properties of carbon nanofiber web as an electrode for

supercapacitor prepared by electrospinning, Applied Physics Letters, 2003, 83 (6): 1216-1218.

Kim, C., Yang, K.S., Kojima, M., Yoshida, K., Kim, Y.J., Kim, Y.A., Endo, M., Fabrication of

Electrospinning-Derived Carbon Nanofiber Webs for the Anode Material of Lithium-Ion

Secondary Batteries, Advanced Functional Materials, 2006, 16 (18): 2393-2397.

Kim, H., Hyun, M., Chang, I., Kim, B., A Microbial Fuel Cell Type Lactate Biosensor Using a Metal -

Reducing Bacterium , Shewanella putrefaciens, Journal of Microbiology and Biotechnology,

1999d, 9 (3): 365-367

Kim, H.J., Park, H.S., Hyun, M.S., Chang, I.S., Kim, M., Kim, B.H., A mediator-less microbial fuel cell

using a metal reducing bacterium, Shewanella putrefaciens, Enzyme and Microbial Technology,

2002, 30 (2): 145-152.

Kim, J.R., Min, B., Logan, B.E., Evaluation of procedures to acclimate a microbial fuel cell for

electricity production, Applied Microbiology and Biotechnology, 2005, 68 (1): 23-30.

Kim, Y., Logan, B.E., Series Assembly of Microbial Desalination Cells Containing Stacked

Electrodialysis Cells for Partial or Complete Seawater Desalination, Environmental Science &

Technology, 2011, 45 (13): 5840-5845.

Kolker, E., Picone, A.F., Galperin, M.Y., Romine, M.F., Higdon, R., Makarova, K.S., Kolker, N.,

Anderson, G.A., Qiu, X., Auberry, K.J., Babnigg, G., Beliaev, A.S., Edlefsen, P., Elias, D.A.,

Gorby, Y.A., Holzman, T., Klappenbach, J.A., Konstantinidis, K.T., Land, M.L., Lipton, M.S.,

McCue, L.A., Monroe, M., Pasa-Tolic, L., Pinchuk, G., Purvine, S., Serres, M.H., Tsapin, S.,

Zakrajsek, B.A., Zhu, W., Zhou, J., Larimer, F.W., Lawrence, C.E., Riley, M., Collart, F.R.,

Yates Iii, J.R., Smith, R.D., Giometti, C.S., Nealson, K.H., Fredrickson, J.K., Tiedje, J.M.,

Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and

improved functional annotations, Proceedings of the National Academy of Sciences of the United

States of America, 2005, 102 (6): 2099-2104.

Koons, B.W., Baeseman, J.L., Novak, P.J., Investigation of cell exudates active in carbon tetrachloride

and chloroform degradation, Biotechnology and Bioengineering, 2001, 74 (1): 12-17.

LaBelle, E., Bond, D.R., Lowy, D.A., Manohar, A.K., He, Z., Mansfeld, F., Electrochemical Techniques

for The Analysis of Bioelectrochemical Systems, in: K. Rabaey, L. Angenent, U. Schroder, J.

Keller (Eds.) Bioelectrochemical Systems: from Extracellular Electron Transfer to

Biotechnological Application

2010, pp. 135-184.

Page 176: PhD Thesis Alessandro Carmona2012

-155-

Lee, H.-S., Parameswaran, P., Kato-Marcus, A., Torres, C.I., Rittmann, B.E., Evaluation of energy-

conversion efficiencies in microbial fuel cells (MFCs) utilizing fermentable and non-fermentable

substrates, Water Research, 2008, 42 (6-7): 1501-1510.

Lewandowski, Z., Beyenal, H., Stookey, D., Reproducibility of biofilm processess and the meaning of

steady state in biofilm reactors, Water Science and Technology, 2004, 49 (11-12): 359-364.

Lewis, K., Symposium on bioelectrochemistry of microorganisms. IV. Biochemical fuel cells,

Bacteriological reviews, 1966, 30 (1): 101-113.

Lewis, T.A., Paszczynski, A., Gordon-Wylie, S.W., Jeedigunta, S., Lee, C.H., Crawford, R.L., Carbon

tetrachloride dechlorination by the bacterial transition metal chelator pyridine-2, 6-bis

(thiocarboxylic acid), Environmental Science & Technology, 2001, 35 (3): 552-559.

Li, S.-L., Freguia, S., Liu, S.-M., Cheng, S.-S., Tsujimura, S., Shirai, O., Kano, K., Effects of oxygen on

Shewanella decolorationis NTOU1 electron transfer to carbon-felt electrodes, Biosensors and

Bioelectronics, 2010, 25 (12): 2651-2656.

Li, X., Li, Y., Li, F., Zhou, S., Feng, C., Liu, T., Interactively interfacial reaction of iron-reducing

bacterium and goethite for reductive dechlorination of chlorinated organic compounds, Chinese

Science Bulletin, 2009a, 54 (16): 2800-2804.

Li, X.M., Zhou, S.G., Li, F.B., Wu, C.Y., Zhuang, L., Xu, W., Liu, L., Fe (III) oxide reduction and

carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17,

Journal of Applied Microbiology, 2009b, 106 (1): 130-139.

Lies, D.P., Hernandez, M.E., Kappler, A., Mielke, R.E., Gralnick, J.A., Newman, D.K., Shewanella

oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct

Contact under Conditions Relevant for Biofilms, Applied and Environmental Microbiology,

2005, 71 (8): 4414-4426.

Liu, H., Cheng, S., Logan, B.E., Production of Electricity from Acetate or Butyrate Using a Single-

Chamber Microbial Fuel Cell, Environmental Science & Technology, 2004, 39 (2): 658-662.

Liu, H., Cheng, S., Logan, B.E., Power generation in fed-batch microbial fuel cells as a function of ionic

strength, temperature, and reactor configuration, Environmental Science and Technology, 2005,

39 (14): 5488-5493.

Liu, H., Matsuda, S., Kato, S., Hashimoto, K., Nakanishi, S., Redox-Responsive Switching in Bacterial

Respiratory Pathways Involving Extracellular Electron Transfer, ChemSusChem, 2010a, 3 (11):

1253-1256.

Liu, H., Matsuda, S., Kawai, T., Hashimoto, K., Nakanishi, S., Feedback stabilization involving redox

states of c-type cytochromes in living bacteria, Chemical Communications, 2011, 47 (13): 3870-

3872.

Liu, M., Yuan, Y., Zhang, L.X., Zhuang, L., Zhou, S.G., Ni, J.R., Bioelectricity generation by a Gram-

positive Corynebacterium sp. strain MFC03 under alkaline condition in microbial fuel cells,

Bioresource Technology, 2010b, 101 (6): 1807-1811.

Liu, X.-c., Zhang, Y., Yang, M., Wang, Z.-y., Lv, W.-z., Analysis of bacterial community structures in

two sewage treatment plants with different sludge properties and treatment performance by

nested PCR-DGGE method, Journal of Environmental Sciences, 2007, 19 (1): 60-66.

Liu, Y., Harnisch, F., Fricke, K., Schröder, U., Climent, V., Feliu, J.M., The study of electrochemically

active microbial biofilms on different carbon-based anode materials in microbial fuel cells,

Biosensors and Bioelectronics, 2010c, 25 (9): 2167-2171.

Liu, Y., Harnisch, F., Fricke, K., Sietmann, R., Schröder, U., Improvement of the anodic

bioelectrocatalytic activity of mixed culture biofilms by a simple consecutive electrochemical

selection procedure, Biosensors and Bioelectronics, 2008, 24 (4): 1006-1011.

Logan, B., Scaling up microbial fuel cells and other bioelectrochemical systems, Applied Microbiology

and Biotechnology, 2010, 85 (6): 1665-1671.

Logan, B., Cheng, S., Watson, V., Estadt, G., Graphite fiber brush anodes for increased power

production in air-cathode microbial fuel cells, Environmental Science and Technology, 2007, 41

(9): 3341-3346.

Logan, B.E., Exoelectrogenic bacteria that power microbial fuel cells, Nature Reviews Microbiology,

2009, 7 (5): 375-381.

Page 177: PhD Thesis Alessandro Carmona2012

-156-

Logan, B.E., Call, D., Cheng, S., Hamelers, H.V.M., Sleutels, T.H.J.A., Jeremiasse, A.W., Rozendal,

R.A., Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic

Matter, Environmental Science & Technology, 2008, 42 (23): 8630-8640.

Logan, B.E., Hamelers, B., Rozendal, R., Schröder, U., Keller, J., Freguia, S., Aelterman, P., Verstraete,

W., Rabaey, K., Microbial Fuel Cells: Methodology and Technology, Environmental Science &

Technology, 2006, 40 (17): 5181-5192.

Logan, B.E., Regan, J.M., Electricity-producing bacterial communities in microbial fuel cells, Trends in

microbiology, 2006a, 14 (12): 512-518.

Logan, B.E., Regan, J.M., Microbial Fuel Cells - Challenges and Applications, Environmental Science &

Technology, 2006b, 40 (17): 5172-5180.

Lovley, D.R., Dissimilatory Fe(III) and Mn(IV) reduction, Microbiological Reviews, 1991, 55 (2): 259-

287.

Lovley, D.R., Dissimilatory metal reduction, Annual Reviews in Microbiology, 1993, 47 (1): 263-290.

Lovley, D.R., Environmental microbe-metal interactions, ASM, 2000.

Lovley, D.R., Bug juice: harvesting electricity with microorganisms, Nat Rev Micro, 2006, 4 (7): 497-

508.

Lovley, D.R., Extracellular electron transfer: wires, capacitors, iron lungs, and more, Geobiology, 2008a,

6 (3): 225-231.

Lovley, D.R., The microbe electric: conversion of organic matter to electricity, Current Opinion in

Biotechnology, 2008b, 19 (6): 564-571.

Lovley, D.R., Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of

energy-related contamination, Energy & Environmental Science, 2011, 4 (12): 4896-4906.

Lovley, D.R., Coates, J.D., Blunt-Harris, E.L., Phillips, E.J.P., Woodward, J.C., Humic substances as

electron acceptors for microbial respiration, Nature, 1996, 382 (6590): 445-448.

Lovley, D.R., Giovannoni, S.J., White, D.C., Champine, J.E., Phillips, E.J.P., Gorby, Y.A., Goodwin, S.,

Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete

oxidation of organic compounds to the reduction of iron and other metals, Archives of

Microbiology, 1993, 159 (4): 336-344.

Lovley, D.R., Phillips, E.J., Lonergan, D.J., Widman, P.K., Fe(III) and S0 reduction by Pelobacter

carbinolicus, Applied and Environmental Microbiology, 1995, 61 (6): 2132-2138.

Lovley, D.R., Phillips, E.J.P., Novel Mode of Microbial Energy Metabolism: Organic Carbon Oxidation

Coupled to Dissimilatory Reduction of Iron or Manganese, Applied and Environmental

Microbiology, 1988, 54 (6): 1472-1480.

Luo, H., Xu, P., Roane, T.M., Jenkins, P.E., Ren, Z., Microbial desalination cells for improved

performance in wastewater treatment, electricity production, and desalination, Bioresource

Technology, 2012, 105 (0): 60-66.

Luu, Y.S., Ramsay, J.A., Review: microbial mechanisms of accessing insoluble Fe (III) as an energy

source, World Journal of Microbiology and Biotechnology, 2003, 19 (2): 215-225.

Malvankar, N.S., Vargas, M., Nevin, K.P., Franks, A.E., Leang, C., Kim, B.-C., Inoue, K., Mester, T.,

Covalla, S.F., Johnson, J.P., Rotello, V.M., Tuominen, M.T., Lovley, D.R., Tunable metallic-like

conductivity in microbial nanowire networks, Nat Nano, 2011, 6 (9): 573-579.

Marshall, C.W., May, H.D., Electrochemical evidence of direct electrode reduction by a thermophilic

Gram-positive bacterium, Thermincola ferriacetica, Energy & Environmental Science, 2009, 2

(6): 699-705.

Marsili, E., Baron, D.B., Shikhare, I.D., Coursolle, D., Gralnick, J.A., Bond, D.R., Shewanella secretes

flavins that mediate extracellular electron transfer, Proceedings of the National Academy of

Sciences, 2008a, 105 (10): 3968-3973.

Marsili, E., Rollefson, J.B., Baron, D.B., Hozalski, R.M., Bond, D.R., Microbial Biofilm Voltammetry:

Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms, Applied and

Environmental Microbiology, 2008b, 74 (23): 7329-7337.

Marsili, E., Sun, J., Bond, D.R., Voltammetry and growth physiology of Geobacter sulfurreducens

biofilms as a function of growth stage and imposed electrode potential, Electroanalysis, 2010, 22

(7-8): 865-874.

Page 178: PhD Thesis Alessandro Carmona2012

-157-

Marsili, E., Zhang, X., Shuttling via soluble compounds, in: K. Rabaey, L. Angenent, U. Schroder, J.

Keller (Eds.) Bioelectrochemical Systems: from Extracellular Electron Transfer to

Biotechnological Application, 2010, pp. 59-80.

Masuda, M., Freguia, S., Wang, Y.-F., Tsujimura, S., Kano, K., Flavins contained in yeast extract are

exploited for anodic electron transfer by Lactococcus lactis, Bioelectrochemistry, 2010, 78 (2):

173-175.

Meitl, L.A., Eggleston, C.M., Colberg, P.J.S., Khare, N., Reardon, C.L., Shi, L., Electrochemical

interaction of Shewanella oneidensis MR-1 and its outer membrane cytochromes OmcA and

MtrC with hematite electrodes, Geochimica et Cosmochimica Acta, 2009, 73 (18): 5292-5307.

Methé, B.A., Nelson, K.E., Eisen, J.A., Paulsen, I.T., Nelson, W., Heidelberg, J.F., Wu, D., Wu, M.,

Ward, N., Beanan, M.J., Dodson, R.J., Madupu, R., Brinkac, L.M., Daugherty, S.C., DeBoy,

R.T., Durkin, A.S., Gwinn, M., Kolonay, J.F., Sullivan, S.A., Haft, D.H., Selengut, J., Davidsen,

T.M., Zafar, N., White, O., Tran, B., Romero, C., Forberger, H.A., Weidman, J., Khouri, H.,

Feldblyum, T.V., Utterback, T.R., Van Aken, S.E., Lovley, D.R., Fraser, C.M., Genome of

Geobacter sulfurreducens: Metal Reduction in Subsurface Environments, Science, 2003, 302

(5652): 1967-1969.

Meyer, T.E., Tsapin, A.I., Vandenberghe, I., De Smet, L., Frishman, D., Nealson, K.H., Cusanovich,

M.A., Van Beeumen, J.J., Identification of 42 Possible Cytochrome C Genes in the Shewanella

oneidensis Genome and Characterization of Six Soluble Cytochromes, OMICS A Journal of

Integrative Biology, 2004, 8 (1): 57-77.

Miller, T.L., Wolin, M.J., A serum bottle modification of the Hungate technique for cultivating obligate

anaerobes, Journal of Applied Microbiology, 1974, 27 (5): 985-987.

Milliken, C., May, H., Sustained generation of electricity by the spore-forming, Gram-positive,

Desulfitobacterium hafniense; strain DCB2, Applied Microbiology and Biotechnology, 2007, 73

(5): 1180-1189.

Millo, D., Harnisch, F., Patil, S.A., Ly, H.K., Schröder, U., Hildebrandt, P., In situ

spectroelectrochemical investigation of electrocatalytic microbial biofilms by surface-enhanced

resonance raman spectroscopy, Angewandte Chemie - International Edition, 2011, 50 (11):

2625-2627.

Min, B., Cheng, S., Logan, B.E., Electricity generation using membrane and salt bridge microbial fuel

cells, Water Research, 2005, 39 (9): 1675-1686.

Min, B., Logan, B.E., Continuous Electricity Generation from Domestic Wastewater and Organic

Substrates in a Flat Plate Microbial Fuel Cell, Environmental Science & Technology, 2004, 38

(21): 5809-5814.

Minteer, S.D., Liaw, B.Y., Cooney, M.J., Enzyme-based biofuel cells, Current Opinion in

Biotechnology, 2007, 18 (3): 228-234.

Müller, S., Bley, T., Berney, M., Blank, L.M.C.O.N., Insight into natural microbial community dynamics

using a combined approach of community fingerprinting, flow cytometry and trend interpretation

analysis in: T. Sheper (Ed.) Advances in Biochemical Engineering and Biotechnology: High

Resolution Microbial Single Cell Analytics, Springer, Berlin, 2011, pp. 233.

Müller, S., Nebe-Von-Caron, G., Functional single-cell analyses: Flow cytometry and cell sorting of

microbial populations and communities, FEMS Microbiology Reviews, 2010, 34 (4): 554-587.

Murray, A.E., Lies, D., Li, G., Nealson, K., Zhou, J., Tiedje, J.M., DNA/DNA hybridization to

microarrays reveals gene-specific differences between closely related microbial genomes,

Proceedings of the National Academy of Sciences, 2001, 98 (17): 9853-9858.

Myers, C.R., Nealson, K.H., Bacterial Manganese Reduction and Growth with Manganese Oxide as the

Sole Electron Acceptor, Science, 1988, 240 (4857): 1319-1321.

Nakamura, R., Ishii, K., Hashimoto, K., Electronic Absorption Spectra and Redox Properties of C Type

Cytochromes in Living Microbes, Angewandte Chemie International Edition, 2009a, 48 (9):

1606-1608.

Nakamura, R., Kai, F., Okamoto, A., Newton, G.J., Hashimoto, K., Self-Constructed Electrically

Conductive Bacterial Networks, Angewandte Chemie International Edition, 2009b, 48 (3): 508-

511.

Nealson, K.H., Myers, C.R., Microbial reduction of manganese and iron: new approaches to carbon

cycling, Applied and Environmental Microbiology, 1992, 58 (2): 439-443.

Page 179: PhD Thesis Alessandro Carmona2012

-158-

Nealson, K.H., Myers, C.R., Wimpee, B.B., Isolation and identification of manganese-reducing bacteria

and estimates of microbial Mn(IV)-reducing potential in the Black Sea, Deep Sea Research Part

A. Oceanographic Research Papers, 1991, 38, Supplement 2 (0): S907-S920.

Nealson, K.H., Saffarini, D., Iron and manganese in anaerobic respiration: environmental significance,

physiology, and regulation, Annual Reviews in Microbiology, 1994, 48 (1): 311-343.

Nealson, K.H., Scott, J., Ecophysiology of the genus Shewanella, The Prokaryotes, 2006, 6: 1133-1151.

Neu, T.R., Manz, B., Volke, F., Dynes, J.J., Hitchcock, A.P., Lawrence, J.R., Advanced imaging

techniques for assessment of structure, composition and function in biofilm systems, FEMS

Microbiology Ecology, 2010, 72 (1): 1-21.

Nevin, K.P., Woodard, T.L., Franks, A.E., Summers, Z.M., Lovley, D.R., Microbial Electrosynthesis:

Feeding Microbes Electricity To Convert Carbon Dioxide and Water to Multicarbon

Extracellular Organic Compounds, mBio, 2010, 1 (2).

Newton, G.J., Mori, S., Nakamura, R., Hashimoto, K., Watanabe, K., Analyses of Current-Generating

Mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in Microbial Fuel

Cells, Applied and Environmental Microbiology, 2009, 75 (24): 7674-7681.

Nielsen, L.P., Risgaard-Petersen, N., Fossing, H., Christensen, P.B., Sayama, M., Electric currents

couple spatially separated biogeochemical processes in marine sediment, Nature, 2010, 463

(7284): 1071-1074.

Nimje, V.R., Chen, C.-Y., Chen, H.-R., Chen, C.-C., Huang, Y.M., Tseng, M.-J., Cheng, K.-C., Chang,

Y.-F., Comparative bioelectricity production from various wastewaters in microbial fuel cells

using mixed cultures and a pure strain of Shewanella oneidensis, Bioresource Technology, 2012,

104 (0): 315-323.

Oellerich, S., Wackerbarth, H., Hildebrandt, P., Spectroscopic Characterization of Nonnative

Conformational States of Cytochrome c, The Journal of Physical Chemistry B, 2002, 106 (25):

6566-6580.

Okamoto, A., Nakamura, R., Hashimoto, K., In-vivo identification of direct electron transfer from

Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein

complexes, Electrochimica Acta, 2011, 56 (16): 5526-5531.

Okamoto, A., Nakamura, R., Ishii , K., Hashimoto , K., In vivo Electrochemistry of C-Type Cytochrome-

Mediated Electron-Transfer with Chemical Marking, ChemBioChem, 2009, 10 (14): 2329-2332.

Owen, R.J., Legros, R.M., Lapage, S.P., Base Composition, Size and Sequence Similarities of Genome

Deoxyribonucleic Acids from Clinical Isolates of Pseudomonas putrefaciens, Journal of General

Microbiology, 1978, 104 (1): 127-138.

Pan, X., Yan, B., Yoh, M., Effects of land use and changes in cover on the transformation and

transportation of iron: A case study of the Sanjiang Plain, Northeast China, Science China Earth

Sciences, 2011, 54 (5): 686-693.

Pankhurst, K.L., Mowat, C.G., Rothery, E.L., Hudson, J.M., Jones, A.K., Miles, C.S., Walkinshaw,

M.D., Armstrong, F.A., Reid, G.A., Chapman, S.K., A Proton Delivery Pathway in the Soluble

Fumarate Reductase from Shewanella frigidimarina, Journal of Biological Chemistry, 2006, 281

(29): 20589-20597.

Pant, D., Van Bogaert, G., Diels, L., Vanbroekhoven, K., A review of the substrates used in microbial

fuel cells (MFCs) for sustainable energy production, Bioresource Technology, 2010, 101 (6):

1533-1543.

Park, Zeikus, Impact of electrode composition on electricity generation in a single-compartment fuel cell

using Shewanella putrefaciens, Applied Microbiology and Biotechnology, 2002, 59 (1): 58-61.

Park, D.H., Zeikus, J.G., Electricity Generation in Microbial Fuel Cells Using Neutral Red as an

Electronophore, Applied and Environmental Microbiology, 2000, 66 (4): 1292-1297.

Park, H.S., Kim, B.H., Kim, H.S., Kim, H.J., Kim, G.T., Kim, M., Chang, I.S., Park, Y.K., Chang, H.I.,

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to

Clostridium butyricum Isolated from a Microbial Fuel Cell, Anaerobe, 2001, 7 (6): 297-306.

Patil, S.A., Harnisch, F., Kapadnis, B., Schröder, U., Electroactive mixed culture biofilms in microbial

bioelectrochemical systems: The role of temperature for biofilm formation and performance,

Biosensors and Bioelectronics, 2010, 26 (2): 803-808.

Patil, S.A., Harnisch, F., Koch, C., Hübschmann, T., Fetzer, I., Carmona-Martínez, A.A., Müller, S.,

Schröder, U., Electroactive mixed culture derived biofilms in microbial bioelectrochemical

Page 180: PhD Thesis Alessandro Carmona2012

-159-

systems: The role of pH on biofilm formation, performance and composition, Bioresource

Technology, 2011, 102 (20): 9683-9690.

Peng, L., You, S.-J., Wang, J.-Y., Carbon nanotubes as electrode modifier promoting direct electron

transfer from Shewanella oneidensis, Biosensors and Bioelectronics, 2010a, 25 (5): 1248-1251.

Peng, L., You, S.-J., Wang, J.-Y., Electrode potential regulates cytochrome accumulation on Shewanella

oneidensis cell surface and the consequence to bioelectrocatalytic current generation, Biosensors

and Bioelectronics, 2010b, 25 (11): 2530-2533.

Pham, C.A., Jung, S.J., Phung, N.T., Lee, J., Chang, I.S., Kim, B.H., Yi, H., Chun, J., A novel

electrochemically active and Fe(III)-reducing bacterium phylogenetically related to Aeromonas

hydrophila, isolated from a microbial fuel cell, FEMS Microbiology Letters, 2003, 223 (1): 129-

134.

Pham, Q.P., Sharma, U., Mikos, A.G., Electrospinning of polymeric nanofibers for tissue engineering

applications: A review, Tissue Engineering, 2006, 12 (5): 1197-1211.

Pham, T., Boon, N., Aelterman, P., Clauwaert, P., De Schamphelaire, L., Vanhaecke, L., De Maeyer, K.,

Höfte, M., Verstraete, W., Rabaey, K., Metabolites produced by Pseudomonas sp. enable a

Gram-positive bacterium to achieve extracellular electron transfer, Applied Microbiology and

Biotechnology, 2008, 77 (5): 1119-1129.

Pisciotta, J., Zou, Y., Baskakov, I., Role of the photosynthetic electron transfer chain in electrogenic

activity of cyanobacteria, Applied Microbiology and Biotechnology, 2011, 91 (2): 377-385.

Pivnick, H., Pseudomonas rubescens, a new species from soluble oil emulsions, Journal of Bacteriology,

1955, 70 (1): 1-6.

Potter, M.C., Electrical Effects Accompanying the Decomposition of Organic Compounds, Proceedings

of the Royal Society of London. Series B, Containing Papers of a Biological Character, 1911, 84

(571): 260-276.

Prasad, D., Arun, S., Murugesan, M., Padmanaban, S., Satyanarayanan, R.S., Berchmans, S.,

Yegnaraman, V., Direct electron transfer with yeast cells and construction of a mediatorless

microbial fuel cell, Biosensors and Bioelectronics, 2007, 22 (11): 2604-2610.

Preciado-Flores, S., Wheeler, D.A., Tran, T.M., Tanaka, Z., Jiang, C., Barboza-Flores, M., Qian, F., Li,

Y., Chen, B., Zhang, J.Z., SERS spectroscopy and SERS imaging of Shewanella oneidensis

using silver nanoparticles and nanowires, Chemical Communications, 2011, 47 (14): 4129-4131.

Price-Whelan, A., Dietrich, L.E.P., Newman, D.K., Rethinking 'secondary' metabolism: physiological

roles for phenazine antibiotics, Nat Chem Biol, 2006, 2 (2): 71-78.

Puig, S., Serra, M., Coma, M., Cabré, M., Balaguer, M.D., Colprim, J., Effect of pH on nutrient

dynamics and electricity production using microbial fuel cells, Bioresource Technology, 2010,

101 (24): 9594-9599.

Qiao, Y., Bao, S.-J., Li, C.M., Cui, X.-Q., Lu, Z.-S., Guo, J., Nanostructured Polyaniline/Titanium

Dioxide Composite Anode for Microbial Fuel Cells, ACS Nano, 2008, 2 (1): 113-119.

Qiao, Y., Li, C.M., Bao, S.-J., Bao, Q.-L., Carbon nanotube/polyaniline composite as anode material for

microbial fuel cells, Journal of Power Sources, 2007, 170 (1): 79-84.

Rabaey, K., Bioelectrochemical Systems: A New Approach Towards Environmental and Industrial

Biotechnology, in: K. Rabaey, L. Angenent, U. Schroder, J. Keller (Eds.) Bioelectrochemical

Systems: from Extracellular Electron Transfer to Biotechnological Application, 2010, pp. 1-16.

Rabaey, K., Angenent, L., Schroder, U., Keller, J., Bioelectrochemical Systems: from Extracellular

Electron Transfer to Biotechnological Application, 2009.

Rabaey, K., Angenent, L., Schröder, U., Keller, J., Bioelectrochemical Systems: from Extracellular

Electron Transfer to Biotechnological Application, IWA Publishing, London, UK, 2010.

Rabaey, K., Boon, N., Höfte, M., Verstraete, W., Microbial phenazine production enhances electron

transfer in biofuel cells, Environmental Science and Technology, 2005, 39 (9): 3401-3408.

Rabaey, K., Boon, N., Siciliano, S.D., Verhaege, M., Verstraete, W., Biofuel cells select for microbial

consortia that self-mediate electron transfer, Applied and Environmental Microbiology, 2004, 70

(9): 5373-5382.

Rabaey, K., Rodriguez, J., Blackall, L.L., Keller, J., Gross, P., Batstone, D., Verstraete, W., Nealson,

K.H., Microbial ecology meets electrochemistry: electricity-driven and driving communities,

ISME J, 2007, 1 (1): 9-18.

Page 181: PhD Thesis Alessandro Carmona2012

-160-

Rabaey, K., Rozendal, R.A., Microbial electrosynthesis - revisiting the electrical route for microbial

production, Nat Rev Micro, 2010, 8 (10): 706-716.

Rabaey, K., Verstraete, W., Microbial fuel cells: novel biotechnology for energy generation, Trends in

Biotechnology, 2005, 23 (6): 291-298.

Rawson, F.J., Garrett, D.J., Leech, D., Downard, A.J., Baronian, K.H.R., Electron transfer from Proteus

vulgaris to a covalently assembled, single walled carbon nanotube electrode functionalised with

osmium bipyridine complex: Application to a whole cell biosensor, Biosensors and

Bioelectronics, 2011, 26 (5): 2383-2389.

Reguera, G., McCarthy, K.D., Mehta, T., Nicoll, J.S., Tuominen, M.T., Lovley, D.R., Extracellular

electron transfer via microbial nanowires, Nature, 2005, 435 (7045): 1098-1101.

Reimers, C.E., Tender, L.M., Fertig, S., Wang, W., Harvesting Energy from the Marine

Sediment−Water Interface, Environmental Science & Technology, 2000, 35 (1): 192-195.

Reneker, D.H., Yarin, A.L., Zussman, E., Xu, H., Electrospinning of Nanofibers from Polymer Solutions

and Melts, in: Advances in Applied Mechanics, 2007, pp. 43-195,345-346.

Richter, H., Nevin, K.P., Jia, H., Lowy, D.A., Lovley, D.R., Tender, L.M., Cyclic voltammetry of

biofilms of wild type and mutant Geobacter sulfurreducens on fuel cell anodes indicates possible

roles of OmcB, OmcZ, type IV pili, and protons in extracellular electron transfer, Energy &

Environmental Science, 2009, 2 (5): 506-516.

Ringeisen, B.R., Henderson, E., Wu, P.K., Pietron, J., Ray, R., Little, B., Biffinger, J.C., Jones-Meehan,

J.M., High power density from a miniature microbial fuel cell using Shewanella oneidensis

DSP10, Environmental Science and Technology, 2006, 40 (8): 2629-2634.

Roden, E.E., Lovley, D.R., Dissimilatory Fe(III) Reduction by the Marine Microorganism

Desulfuromonas acetoxidans, Applied and Environmental Microbiology, 1993, 59 (3): 734-742.

Rollefson, J.B., Levar, C.E., Bond, D.R., Identification of Genes Involved in Biofilm Formation and

Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens, Journal of

Bacteriology, 2009, 191 (13): 4207-4217.

Rosenbaum, M., Aulenta, F., Villano, M., Angenent, L.T., Cathodes as electron donors for microbial

metabolism: Which extracellular electron transfer mechanisms are involved?, Bioresource

Technology, 2011, 102 (1): 324-333.

Rosenbaum, M., Cotta, M.A., Angenent, L.T., Aerated Shewanella oneidensis in continuously fed

bioelectrochemical systems for power and hydrogen production, Biotechnology and

Bioengineering, 2010a, 105 (5): 880-888.

Rosenbaum, M., He, Z., Angenent, L.T., Light energy to bioelectricity: photosynthetic microbial fuel

cells, Current Opinion in Biotechnology, 2010b, 21 (3): 259-264.

Rosenbaum, M., Schröder, U., Photomicrobial Solar and Fuel Cells, Electroanalysis, 2010, 22 (7-8):

844-855.

Rosenbaum, M., Zhao, F., Schröder, U., Scholz, F., Interfacing electrocatalysis and biocatalysis with

tungsten carbide: A high-performance, noble-metal-free microbial fuel cell, Angewandte Chemie

- International Edition, 2006, 45 (40): 6658-6661.

Ross, D.E., Flynn, J.M., Baron, D.B., Gralnick, J.A., Bond, D.R., Towards Electrosynthesis in

Shewanella: Energetics of Reversing the Mtr Pathway for Reductive Metabolism, PLoS ONE,

2011, 6 (2): e16649.

Rozendal, R.A., Hamelers, H.V.M., Buisman, C.J.N., Effects of membrane cation transport on pH and

microbial fuel cell performance, Environmental Science and Technology, 2006a, 40 (17): 5206-

5211.

Rozendal, R.A., Hamelers, H.V.M., Euverink, G.J.W., Metz, S.J., Buisman, C.J.N., Principle and

perspectives of hydrogen production through biocatalyzed electrolysis, International Journal of

Hydrogen Energy, 2006b, 31 (12): 1632-1640.

Rozendal, R.A., Hamelers, H.V.M., Rabaey, K., Keller, J., Buisman, C.J.N., Towards practical

implementation of bioelectrochemical wastewater treatment, Trends in Biotechnology, 2008, 26

(8): 450-459.

Schroder, U., Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency,

Physical Chemistry Chemical Physics, 2007, 9 (21): 2619-2629.

Schröder, U., Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency,

Physical Chemistry Chemical Physics, 2007, 9 (21): 2619-2629.

Page 182: PhD Thesis Alessandro Carmona2012

-161-

Schröder, U., From Wastewater to Hydrogen: Biorefineries Based on Microbial Fuel-Cell Technology,

ChemSusChem, 2008, 1 (4): 281-282.

Schröder, U., Discover the possibilities: microbial bioelectrochemical systems and the revival of a 100-

year–old discovery, Journal of Solid State Electrochemistry, 2011, 15 (7): 1481-1486.

Schröder, U., Harnisch, F., Electrochemical Losses, in: K. Rabaey, L. Angenent, U. Schroder, J. Keller

(Eds.) Bioelectrochemical Systems: from Extracellular Electron Transfer to Biotechnological

Application, 2010, pp. 119-134.

Schröder, U., Nießen, J., Scholz, F., A Generation of Microbial Fuel Cells with Current Outputs Boosted

by More Than One Order of Magnitude, Angewandte Chemie International Edition, 2003, 42

(25): 2880-2883.

Scott, K., Rimbu, G.A., Katuri, K.P., Prasad, K.K., Head, I.M., Application of modified carbon anodes in

microbial fuel cells, Process Safety and Environmental Protection, 2007, 85 (5 B): 481-488.

Šefčovičová, J., Filip, J., Gemeiner, P., Vikartovská, A., Pätoprstý, V., Tkac, J., High performance

microbial 3-D bionanocomposite as a bioanode for a mediated biosensor device,

Electrochemistry Communications, 2011, 13 (9): 966-968.

Sharma, Y., Li, B., The variation of power generation with organic substrates in single-chamber

microbial fuel cells (SCMFCs), Bioresource Technology, 2010, 101 (6): 1844-1850.

Shi, L., Chen, B., Wang, Z., Elias, D.A., Mayer, M.U., Gorby, Y.A., Ni, S., Lower, B.H., Kennedy,

D.W., Wunschel, D.S., Mottaz, H.M., Marshall, M.J., Hill, E.A., Beliaev, A.S., Zachara, J.M.,

Fredrickson, J.K., Squier, T.C., Isolation of a High-Affinity Functional Protein Complex

between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of Shewanella

oneidensis MR-1, Journal of Bacteriology, 2006, 188 (13): 4705-4714.

Shi, L., Richardson, D.J., Wang, Z., Kerisit, S.N., Rosso, K.M., Zachara, J.M., Fredrickson, J.K., The

roles of outer membrane cytochromes of Shewanella and Geobacter in extracellular electron

transfer, Environmental Microbiology Reports, 2009, 1 (4): 220-227.

Siegumfeldt, H., Rechinger, K.B., Jakobsen, M., Dynamic changes of intracellular pH in individual lactic

acid bacterium cells in response to a rapid drop in extracellular pH, Applied and Environmental

Microbiology, 2000, 66 (6): 2330-2335.

Sinha-Ray, S., Yarin, A.L., Pourdeyhimi, B., The production of 100/400 nm inner/outer diameter carbon

tubes by solution blowing and carbonization of core-shell nanofibers, Carbon, 2010, 48 (12):

3575-3578.

Srikanth, S., Marsili, E., Flickinger, M.C., Bond, D.R., Electrochemical characterization of Geobacter

sulfurreducens cells immobilized on graphite paper electrodes, Biotechnology and

Bioengineering, 2008, 99 (5): 1065-1073.

Srikar, R., Yarin, A.L., Megaridis, C.M., Bazilevsky, A.V., Kelley, E., Desorption-limited mechanism of

release from polymer nanofibers, Langmuir, 2008, 24 (3): 965-974.

Staudt, C., Horn, H., Hempel, D.C., Neu, T.R., Volumetric measurements of bacterial cells and

extracellular polymeric substance glycoconjugates in biofilms, Biotechnology and

Bioengineering, 2004, 88 (5): 585-592.

Straub, K.L., Kappler, A., Schink, B., Enrichment and isolation of ferric-iron-and humic-acid-reducing

bacteria, Methods in enzymology, 2005, 397: 58-77.

Straub, K.L., Schink, B., Evaluation of electron-shuttling compounds in microbial ferric iron reduction,

FEMS Microbiology Letters, 2003, 220 (2): 229-233.

Strycharz, S.M., Malanoski, A.P., Snider, R.M., Yi, H., Lovley, D.R., Tender, L.M., Application of

cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified

anodes of Geobacter sulfurreducens strain DL1 vs. variant strain KN400, Energy &

Environmental Science, 2011, 4 (3): 896-913.

Sun, M., Zhang, F., Tong, Z.-H., Sheng, G.-P., Chen, Y.-Z., Zhao, Y., Chen, Y.-P., Zhou, S.-Y., Liu, G.,

Tian, Y.-C., Yu, H.-Q., A gold-sputtered carbon paper as an anode for improved electricity

generation from a microbial fuel cell inoculated with Shewanella oneidensis MR-1, Biosensors

and Bioelectronics, 2010, 26 (2): 338-343.

Sund, C., McMasters, S., Crittenden, S., Harrell, L., Sumner, J., Effect of electron mediators on current

generation and fermentation in a microbial fuel cell, Applied Microbiology and Biotechnology,

2007, 76 (3): 561-568.

Page 183: PhD Thesis Alessandro Carmona2012

-162-

Tanaka, K., Kashiwagi, N., Ogawa, T., Effects of light on the electrical output of bioelectrochemical

fuel-cells containing Anabaena variabilis M-2: mechanism of the post-illumination burst,

Journal of Chemical Technology & Biotechnology, 1988, 42 (3): 235-240.

Teo, W.E., Liao, S., Chan, C.K., Ramakrishna, S., Remodeling of three-dimensional hierarchically

organized nanofibrous assemblies, Current Nanoscience, 2008, 4 (4): 361-369.

Thavasi, V., Singh, G., Ramakrishna, S., Electrospun nanofibers in energy and environmental

applications, Energy & Environmental Science, 2008, 1 (2): 205-221.

Thormann, K.M., Saville, R.M., Shukla, S., Pelletier, D.A., Spormann, A.M., Initial Phases of Biofilm

Formation in Shewanella oneidensis MR-1, J. Bacteriol., 2004, 186 (23): 8096-8104.

Thrash, J.C., Coates, J.D., Review: Direct and Indirect Electrical Stimulation of Microbial Metabolism,

Environmental Science & Technology, 2008, 42 (11): 3921-3931.

Thygesen, A., Poulsen, F.W., Min, B., Angelidaki, I., Thomsen, A.B., The effect of different substrates

and humic acid on power generation in microbial fuel cell operation, Bioresource Technology,

2009, 100 (3): 1186-1191.

Torres, C.I., Krajmalnik-Brown, R., Parameswaran, P., Marcus, A.K., Wanger, G., Gorby, Y.A.,

Rittmann, B.E., Selecting anode-respiring bacteria based on anode potential: Phylogenetic,

electrochemical, and microscopic characterization, Environmental Science and Technology,

2009, 43 (24): 9519-9524.

Torres, C.I., Marcus, A.K., Lee, H.S., Parameswaran, P., Krajmalnik-Brown, R., Rittmann, B.E., A

kinetic perspective on extracellular electron transfer by anode-respiring bacteria, FEMS

Microbiology Reviews, 2010, 34 (1): 3-17.

Torres, C.I., Marcus, A.K., Rittmann, B.E., Proton transport inside the biofilm limits electrical current

generation by anode-respiring bacteria, Biotechnology and Bioengineering, 2008, 100 (5): 872-

881.

Turick, C.E., Beliaev, A.S., Zakrajsek, B.A., Reardon, C.L., Lowy, D.A., Poppy, T.E., Maloney, A.,

Ekechukwu, A.A., The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-

phase electron transfer by Shewanella oneidensis MR-1, FEMS Microbiology Ecology, 2009, 68

(2): 223-225.

Turick, C.E., Tisa, L.S., Caccavo, J.F., Melanin Production and Use as a Soluble Electron Shuttle for

Fe(III) Oxide Reduction and as a Terminal Electron Acceptor by Shewanella algae BrY, Applied

and Environmental Microbiology, 2002, 68 (5): 2436-2444.

Turner, K.L., Doherty, M.K., Heering, H.A., Armstrong, F.A., Reid, G.A., Chapman, S.K., Redox

Properties of Flavocytochrome c3 from Shewanella frigidimarina NCIMB400 Biochemistry,

1999, 38 (11): 3302-3309.

Van Gremberghe, I., Vanormelingen, P., Van Der Gucht, K., Souffreau, C., Vyverman, W., De Meester,

L., Priority effects in experimental populations of the cyanobacterium Microcystis,

Environmental Microbiology, 2009, 11 (10): 2564-2573.

van Rij, E.T., Wesselink, M., Chin-A-Woeng, T.F.C., Bloemberg, G.V., Lugtenberg, B.J.J., Influence of

Environmental Conditions on the Production of Phenazine-1-Carboxamide by Pseudomonas

chlororaphis PCL1391, Molecular Plant-Microbe Interactions, 2004, 17 (5): 557-566.

Velasquez-Orta, S.B., Curtis, T.P., Logan, B.E., Energy from algae using microbial fuel cells,

Biotechnology and Bioengineering, 2009, 103 (6): 1068-1076.

Velasquez-Orta, S.B., Head, I.M., Curtis, T.P., Scott, K., Lloyd, J.R., Von Canstein, H., The effect of

flavin electron shuttles in microbial fuel cells current production, Applied Microbiology and

Biotechnology, 2010, 85 (5): 1373-1381.

Venkataraman, A., Rosenbaum, M.A., Perkins, S.D., Werner, J.J., Angenent, L.T., Metabolite-based

mutualism between Pseudomonas aeruginosa PA14 and Enterobacter aerogenes enhances current

generation in bioelectrochemical systems, Energy & Environmental Science, 2011, 4 (11): 4550-

4559.

Venkateswaran, K., Dollhopf, M.E., Aller, R., Stackebrandt, E., Nealson, K.H., Shewanella amazonensis

sp. nov., a novel metal-reducing facultative anaerobe from Amazonian shelf muds, International

Journal of Systematic Bacteriology, 1998, 48 (3): 965-972.

Venkateswaran, K., Moser, D.P., Dollhopf, M.E., Lies, D.P., Saffarini, D.A., MacGregor, B.J.,

Ringelberg, D.B., White, D.C., Nishijima, M., Sano, H., Burghardt, J., Stackebrandt, E.,

Page 184: PhD Thesis Alessandro Carmona2012

-163-

Nealson, K.H., Polyphasic taxonomy of the genus Shewanella and description of Shewanella

oneidensis sp. nov, International Journal of Systematic Bacteriology, 1999, 49 (2): 705-724.

Vincent, K.A., Parkin, A., Armstrong, F.A., Investigating and exploiting the electrocatalytic properties of

hydrogenases, Chemical Reviews, 2007, 107 (10): 4366-4413.

Vogel, B.F., Jørgensen, K., Christensen, H., Olsen, J.E., Gram, L., Differentiation of Shewanella

putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping,

phenotypic characterization, and 16S rRNA gene sequence analysis, Applied and Environmental

Microbiology, 1997, 63 (6): 2189-2199.

von Canstein, H., Ogawa, J., Shimizu, S., Lloyd, J.R., Secretion of flavins by Shewanella species and

their role in extracellular electron transfer, Applied and Environmental Microbiology, 2008, 74

(3): 615-623.

Wackerbarth, H., Klar, U., Gunther, W., Hildebrandt, P., Novel Time-Resolved Surface-Enhanced

(Resonance) Raman Spectroscopic Technique for Studying the Dynamics of Interfacial

Processes: Application to the Electron Transfer Reaction of Cytochrome c at a Silver Electrode,

Appl. Spectrosc., 1999, 53 (3): 283-291.

Wagner, M., Loy, A., Nogueira, R., Purkhold, U., Lee, N., Daims, H., Microbial community composition

and function in wastewater treatment plants, Antonie van Leeuwenhoek, 2002, 81 (1): 665-680.

Wang, B., Wang, Y., Yin, T., Yu, Q., Applications of electrospinning technique in drug delivery,

Chemical Engineering Communications, 2010, 197 (10): 1315-1338.

Wang, J., Li, M., Shi, Z., Li, N., Gu, Z., Direct Electrochemistry of Cytochrome c at a Glassy Carbon

Electrode Modified with Single-Wall Carbon Nanotubes, Analytical Chemistry, 2002a, 74 (9):

1993-1997.

Wang, L., Wang, E., Direct electron transfer between cytochrome c and a gold nanoparticles modified

electrode, Electrochemistry Communications, 2004, 6 (1): 49-54.

Wang, X., Cheng, S., Feng, Y., Merrill, M.D., Saito, T., Logan, B.E., Use of carbon mesh anodes and the

effect of different pretreatment methods on power production in microbial fuel cells,

Environmental Science and Technology, 2009, 43 (17): 6870-6874.

Wang, Y., Serrano, S., Santiago-Aviles, J.J., Conductivity measurement of electrospun PAN-based

carbon nanofiber, Journal of Materials Science Letters, 2002b, 21 (13): 1055-1057.

Ward, M.J., Fu, Q.S., Rhoads, K.R., Yeung, C.H.J., Spormann, A.M., Criddle, C.S., A derivative of the

menaquinone precursor 1, 4-dihydroxy-2-naphthoate is involved in the reductive transformation

of carbon tetrachloride by aerobically grown Shewanella oneidensis MR-1, Applied

Microbiology and Biotechnology, 2004, 63 (5): 571-577.

Watanabe, K., Manefield, M., Lee, M., Kouzuma, A., Electron shuttles in biotechnology, Current

Opinion in Biotechnology, 2009, 20 (6): 633-641.

Wei, J., Liang, P., Huang, X., Recent progress in electrodes for microbial fuel cells, Bioresource

Technology, 2011, 102 (20): 9335-9344.

Wigginton, N.S., Rosso, K.M., Hochella Jr, M.F., Mechanisms of electron transfer in two decaheme

cytochromes from a metal-reducing bacterium, Journal of Physical Chemistry B, 2007, 111 (44):

12857-12864.

Williams, K.H., Nevin, K.P., Franks, A., Englert, A., Long, P.E., Lovley, D.R., Electrode-Based

Approach for Monitoring In Situ Microbial Activity During Subsurface Bioremediation,

Environmental Science & Technology, 2009, 44 (1): 47-54.

Wise, J.K., Yarin, A.L., Megaridis, C.M., Cho, M., Chondrogenic differentiation of human mesenchymal

stem cells on oriented nanofibrous scaffolds: Engineering the superficial zone of articular

cartilage, Tissue Engineering - Part A, 2009, 15 (4): 913-921.

Workman, D.J., Woods, S.L., Gorby, Y.A., Fredrickson, J.K., Truex, M.J., Microbial reduction of

vitamin B12 by Shewanella alga strain BrY with subsequent transformation of carbon

tetrachloride, Environmental Science & Technology, 1997, 31 (8): 2292-2297.

Wrighton, K.C., Thrash, J.C., Melnyk, R.A., Bigi, J.P., Byrne-Bailey, K.G., Remis, J.P., Schichnes, D.,

Auer, M., Chang, C.J., Coates, J.D., Evidence for Direct Electron Transfer by a Gram-Positive

Bacterium Isolated from a Microbial Fuel Cell, Applied and Environmental Microbiology, 2011,

77 (21): 7633-7639.

Page 185: PhD Thesis Alessandro Carmona2012

-164-

Wu, W., Bai, L., Liu, X., Tang, Z., Gu, Z., Nanograss array boron-doped diamond electrode for enhanced

electron transfer from Shewanella loihica PV-4, Electrochemistry Communications, 2011, 13 (8):

872-874.

Xie, X., Ye, M., Hu, L., Liu, N., McDonough, J.R., Chen, W., Alshareef, H.N., Criddle, C.S., Cui, Y.,

Carbon nanotube-coated macroporous sponge for microbial fuel cell electrodes, Energy &

Environmental Science, 2011, 5 (1): 5265-5270.

Xing, D., Zuo, Y., Cheng, S., Regan, J.M., Logan, B.E., Electricity Generation by Rhodopseudomonas

palustris DX-1, Environmental Science & Technology, 2008, 42 (11): 4146-4151.

Xiong, Y., Shi, L., Chen, B., Mayer, M.U., Lower, B.H., Londer, Y., Bose, S., Hochella, M.F.,

Fredrickson, J.K., Squier, T.C., High-affinity binding and direct electron transfer to solid metals

by the Shewanella oneidensis MR-1 Outer membrane c-type cytochrome OmcA, Journal of the

American Chemical Society, 2006, 128 (43): 13978-13979.

Yang, X., Shah, J.D., Wang, H., Nanofiber enabled layer-by-layer approach toward three-dimensional

tissue formation, Tissue Engineering - Part A, 2009, 15 (4): 945-956.

Yang, Y., Sun, G., Guo, J., Xu, M., Differential biofilms characteristics of Shewanella decolorationis

microbial fuel cells under open and closed circuit conditions, Bioresource Technology, 2011, 102

(14): 7093-7098.

You , S.-J., Wang, J.-Y., Ren, N.-Q., Wang, X.-H., Zhang, J.-N., Sustainable Conversion of Glucose into

Hydrogen Peroxide in a Solid Polymer Electrolyte Microbial Fuel Cell, ChemSusChem, 2010, 3

(3): 334-338.

Yu, Y.-Y., Chen, H.-l., Yong, Y.-C., Kim, D.-H., Song, H., Conductive artificial biofilm dramatically

enhances bioelectricity production in Shewanella-inoculated microbial fuel cells, Chemical

Communications, 2011, 47 (48): 12825-12827.

Yuan, Y., Zhou, S., Xu, N., Zhuang, L., Electrochemical characterization of anodic biofilms enriched

with glucose and acetate in single-chamber microbial fuel cells, Colloids and Surfaces B:

Biointerfaces, 2011, 82 (2): 641-646.

Zhang, L., Zhou, S., Zhuang, L., Li, W., Zhang, J., Lu, N., Deng, L., Microbial fuel cell based on

Klebsiella pneumoniae biofilm, Electrochemistry Communications, 2008, 10 (10): 1641-1643.

Zhang, L., Zhu, X., Li, J., Liao, Q., Ye, D., Biofilm formation and electricity generation of a microbial

fuel cell started up under different external resistances, Journal of Power Sources, 2011, 196

(15): 6029-6035.

Zhao, F., Harnisch, F., Schröder, U., Scholz, F., Bogdanoff, P., Herrmann, I., Challenges and constraints

of using oxygen cathodes in microbial fuel cells, Environmental Science and Technology, 2006,

40 (17): 5193-5199.

Zhao, Y., Watanabe, K., Nakamura, R., Mori, S., Liu, H., Ishii, K., Hashimoto, K., Three-Dimensional

Conductive Nanowire Networks for Maximizing Anode Performance in Microbial Fuel Cells,

Chemistry – A European Journal, 2010a, 16 (17): 4982-4985.

Zhao, Y., Watanabe, K., Nakamura, R., Mori, S., Liu, H., Ishii, K., Hashimoto, K., Three-dimensional

conductive nanowire networks for maximizing anode performance in microbial fuel cells,

Chemistry - A European Journal, 2010b, 16 (17): 4982-4985.

Zhou, Z., Lai, C., Zhang, L., Qian, Y., Hou, H., Reneker, D.H., Fong, H., Development of carbon

nanofibers from aligned electrospun polyacrylonitrile nanofiber bundles and characterization of

their microstructural, electrical, and mechanical properties, Polymer, 2009, 50 (13): 2999-3006.

Zuo, Y., Maness, P.-C., Logan, B.E., Electricity Production from Steam-Exploded Corn Stover Biomass,

Energy & Fuels, 2006, 20 (4): 1716-1721.

Zussman, E., Chen, X., Ding, W., Calabri, L., Dikin, D.A., Quintana, J.P., Ruoff, R.S., Mechanical and

structural characterization of electrospun PAN-derived carbon nanofibers, Carbon, 2005, 43

(10): 2175-2185.

Page 186: PhD Thesis Alessandro Carmona2012

Curriculum Vitae

-A-

Alessandro A. Carmona-Martínez

Hagenring 30

38106 Braunschweig

Germany

Born August 19th 1982 Oaxaca, Mexico.

Telephone work: +49 531-391-8429

Mobile: +49 176-8604-6409

E-mail: [email protected], [email protected]

Career Objective

To seek a position in an academic or research institution that encourages innovative research and

promotes intellectual growth by utilizing my expertise and skills.

Education

2008-2012: Ph.D. (Dr. rer. nat.) in Biotechnology candidate (early 2012)

Institute of Environmental and Sustainable Chemistry. Group of Sustainable

Chemistry and Energy Research. Technical University of Braunschweig. Germany.

Thesis: “Study of the extracellular electron transfer processes between Shewanella

strains and electrode materials in bioelectrochemical systems”.

2005-2007: Master of Science in Environmental Biotechnology

Department of Biotechnology and Bioengineering. Centre for Research and Advanced

Studies of the National Polytechnic Institute. Mexico.

Thesis: “Electricity production in a microbial fuel cell fed with spent organic extracts

from hydrogenogenic fermentation of organic solid wastes”

2001-2005: Bachelor of Science in Environmental Engineering.

Interdisciplinary Professional Unit of Biotechnology of the National Polytechnic

Institute. Mexico.

Thesis: “Batch bio-hydrogen production with inhibited methanogenic consortia from

organic solid waste: effect of incubation temperature”

1997-2000: High school education, graduated in Mathematics and Sciences.

Page 187: PhD Thesis Alessandro Carmona2012

Curriculum Vitae

-B-

Peer-reviewed publications

1. 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).

2. A.A. Carmona-Martinez, F. Harnisch, U. Kuhlicke, T.R. Neu, U. Schröder, Electron transfer

and biofilm formation of Shewanella putrefaciens as function of anode potential,

Bioelectrochemistry, Accepted (2012).

3. 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.

4. S. Chen, H. Hou, F. Harnisch, S.A. Patil, A.A. Carmona-Martinez, 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 and Environmental Science, 4 (2011) 1417-1421.

5. S. Chen, G. He, A.A. Carmona-Martinez, 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.

6. 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.

7. H.M. Poggi-Varaldo, A. Carmona-Martínez, A.L. Vázquez-Larios, O. Solorza-Feria, Effect

of inoculum type on the performance of a microbial fuel cell fed with spent organic extracts

from hydrogenogenic fermentation of organic solid wastes, Journal of New Materials for

Electrochemical Systems, 12 (2009) 49-54.

8. I. Valdez-Vazquez, E. Ríos-Leal, K.M. Muñoz-Páez, A. Carmona-Martínez, H.M. Poggi-

Varaldo, Effect of inhibition treatment, type of inocula, and incubation temperature on batch

H2 production from organic solid waste, Biotechnology and Bioengineering, 95 (2006) 342-

349.

9. I. Valdez-Vazquez, E. Ríos-Leal, A. Carmona-Martínez, K.M. Muñoz-Páez, H.M. Poggi-

Varaldo, Improvement of biohydrogen production from solid wastes by intermittent venting

and gas flushing of batch reactors headspace, Environmental Science and Technology, 40

(2006) 3409-3415.

Page 188: PhD Thesis Alessandro Carmona2012

Curriculum Vitae

-C-

Oral/poster presentations in international conferences

1. 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.

2. 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, Ruhr-

Universität Bochum (Germany), September 13th – 15th, 2010.

3. 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.

4. A.A. Carmona-Martínez, 2009. Microbial fuel cells: an alternative for the production of

clean electricity. Abstract F128. Presented at German Academic Exchange Service Scholarship

Holders Meeting. Hanover (Germany). June 19th – 21th, 2009.

5. A. Carmona-Martinez, O. Solorza-Feria, H. M. Poggi-Varaldo. 2008. Batch tests of a

microbial fuel cell for electricity generation from spent organic extracts from hydrogenogenic

fermentation of organic solid wastes**. Abstract 2894. Presented at Third International Meeting on

Environmental Biotechnology and Engineering. Palma de Mallorca (Spain). September 21st - 25th

2008. ISBN: 978-84-692-4948-2.

6. A. A. Carmona-Martínez, O. Solorza-Feria, H. M. Poggi-Varaldo. 2008. Design and

characterization of a microbial fuel cell for electricity production from leachates**. Paper S001.

Presented at Sixth International Conference on Remediation of Chlorinated and Recalcitrant

Compounds. Monterey, California (USA). May 19th – 22th 2008. ISBN: 1-57477-163-9.

7. A. A. Carmona-Martínez, F. Esparza-García, J. García-Mena, O. Solorza-Feria, H. M.

Poggi-Varaldo. 2006. Actualidad y perspectivas en celdas de combustible microbianas para la

obtención de energía eléctrica a partir de residuales. Paper 109. Presented at Second International

Meeting on Environmental Biotechnology and Engineering. Mexico City (Mexico). September

26th – 29th 2006. ISBN: 970-95106-0-6.

**Presented at the congress by Dr. Héctor M. Poggi-Varaldo in my behalf.

Page 189: PhD Thesis Alessandro Carmona2012

Curriculum Vitae

-D-

Work and Research Experience

2012-2013: Coupling hydrogen production by dark fermentation and microbial electrolysis in a

single anaerobic reactor. References: Dr. Nicolas Bernet and Dr. E. Trably at the

Laboratory of Environmental Biotechnology of the French National Institute for

Agricultural Research.

2012: In vivo study of outer membrane cytochromes embedded in aggregations of living

bacteria (i.e microbial biofilms) grown on electrodes by a combination of surface-

enhanced resonance Raman scattering spectroscopy and electrochemistry. Reference:

Dr. Diego Millo at the Chemistry department/ Vrije Universiteit Amsterdam, the

Netherlands.

2008-2011: Experience on Bioelectrochemical systems (BES) aspects such as the extracellular

electron transfer mechanisms between bacteria and electrode materials in microbial

biofilms, analysis of environmental conditions affecting the performing of BES, study

of diverse electrode materials to enhance the performance of microbial biofilms in

microbial fuel cell systems, etc. References: Prof. Dr. Uwe Schröder and Dr. Falk

Harnisch at the Institute of Environmental and Sustainable Chemistry/ Technische

Universität Braunschweig, Germany.

2003-2007: Renewable biofuels (H2 and Biogas) production trough feasible and environmentally

friendly biotechnological process: e.g., hydrogen production from inhibited

methanogenic consortia. Reference: Dr. Héctor M. Poggi-Varaldo from the

Environmental biotechnology laboratory at the Centro de Investigación y de Estudios

Avanzados del Instituto Politécnico Nacional (Cinvestav), Mexico.

2005-2007: Design, construction and characterization of microbial fuel cells. Reference: Dr. Omar

Solorza-Feria at the Hydrogen and fuel cells laboratory, Cinvestav.

2003-2007: Use of analytical techniques for the detection of biotechnological compounds trough

methodologies based on gas and liquid chromatography. Reference: Mrs. Elvira Ríos-

Leal at the Analytic chemistry in biotechnology, Cinvestav.

2006-2007: Experience in the use of molecular tools for genetic typification of the microbioma

and microbial diversity in environmental and biotechnological systems. Reference: Dr.

Jaime García-Mena at the Laboratory of environmental genomics, Cinvestav.

Page 190: PhD Thesis Alessandro Carmona2012

Curriculum Vitae

-E-

Awards and Honours

2012: Foundation Caesar grant for post-doctoral research at the VU Amsterdam.

2008-2011: Ph. D. scholarship by the German Academic Exchange Service (DAAD).

2008-2011: Ph. D. complementary scholarship program by the Secretariat of Public Education of

Mexico (SEP).

2008: Winner in the student paper competition at the Sixth International Conference on

Remediation of Chlorinated & Recalcitrant Compounds (Monterey, California, USA)

2006: Winner of the poster competition in the renewable energies area at the Second

International Meeting on Environmental Biotechnology and Engineering (Mexico

City, Mexico).

2005-2007: M. Sc. scholarship by National Council of Science and Technology (CONACyT).

Professional memberships

-Mexican Talent Network e.V., Germany

-Mexican Society of Biotechnology and Bioengineering, A.C.

-Mexican Society of Hydrogen, A.C.

Linguistic skills Reading Writing Speaking

Spanish Mother tongue Mother tongue Mother tongue

English Proficient Proficient Proficient

German Good Good Good