From Mud

to Electrode Catalysts and

Conductive Nanomaterials
Leonard M. Tender Naval Research Laboratory
Center for Bio/Molecular Science and Engineering, Washington, DC
20375 USA

December 10, 2014

Electrode-grown Geobacter sulfurreducens biofilm

Image: Bond Lab, Univ of Minn

Iron-Reducing Bacteria (FeRB)

Geobacter metallireducens
Image: Lovley Lab, UMass

Shi L, et al. (2012) Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella oneidensis MR-1. Frontiers in microbiology 3.

Bioelectrochemical Systems (BES)

Microbial Electrochemistry/Electro-Microbiology
Micobial Biocathodes

Microbial Bioanodes
Bacteria generate electrons
Bacteria transfer electrons onto electrodes
Bio-anode can be a single species
Anaerobic

CO2
Geobacter
Shewanella
mixed communities
FeRB

e-

Demonstrated bio-cathodes:
Bioremediation
Denitrification
Oxygen reduction
CO2 fixation

e-

Cathode

C2HO

e-

Anode

Demonstrated bio-anodes:
Organic matter oxidation
(pure/sediments/wastes)

Bacteria consume electrons.

Bacteria need carbon CO2.
Cathode needs a consortium.
Bio-cathode consortia/synergy

e-

O2, CO2
Biocathdode MCL
(seawater enriched)

H2O, C2HO
Mariprofundus ferrooxydans
mixed communities
FeOB

Depth-dependent sediment potential gradient assay of marine sediment microbial activity

-0.2 V

water

vs. SHE

0.6 V

sediment

1 - 10 cm

Pt microelectrode
w/insulating sheath
CBMSE 6.2 Review, June 18, 2003
Limmol. Oceanogr. 1969, 14, 547-558

Benthic Redox Gradient

organic matter, oxidants

(CH2O)106(NH3)16(HPO4) + 138O2
106CO2 + 16HNO3 + H3PO4 + 122H2O

Oxygen:

1 - 10 cm

Manganese:

DGo (kJ/mole C6H12O6 )

-181

(CH2O)106(NH3)16(HPO4) + 236MNO2 + 472H+

236Mn2+ + 106CO2 + 8N2 + H3PO4 + 366H2O

-175

Nitrate:

(CH2O)106(NH3)16(HPO4) + 84.4HNO3
106CO2 + 44.2N2 + H3PO4 + 148.4H2O + 16NH3

-156

Iron:

(CH2O)106(NH3)16(HPO4) + 424Fe2O3 + 484H+

424Fe2+ + 106CO2 + 16NH3 + H3PO4 + 742H2O

-24

Sulfate:

(CH2O)106(NH3)16(HPO4) + 53SO42-
106CO2 + 16NH3 + H3PO4 + 53S2- + 106H2O

-21

Methanogenesis:

(CH2O)106(NH3)16(HPO4)
53CO2 CBMSE
+ 53CH
+ H3PO4
4 + 16NH
6.2 Review,
June 18,3 2003

-20

Geocheim, Cosochim. Acta 1979, 43, 1075-1080

A fuel cell? Why?

O2 + H+

H2O

Cathode

eOxic
Water
Anoxic
Sediment

H+

aerobes
O2 + C6H12O6

e-

CO2 + H2O

eAnode
XH

H+ + X-

Voltage and power density vs. current density for Identical

Pt Mesh-based fuel cells in different sediments, laboratory studies
1.2

0.5

1
0.4
0.8
0.3
0.6
0.2

low voltage
ohmic

0.1

0.4
0.2

0
0

Marine sediment &

seawater from a salt
marsh near
Tuckerton, New
Jersey USA,
39o 30.5 N, 74o19.6
W.
(5.5% Reduced
Carbon)

2000

4000

Depth in Sediment (cm)

1.4

~1.2 mWatt/m2

0.6

Voltage (V)

Concentration (mM)

1.6
Power Density (mWatt / m )

0.7

1
HS-

2
3
4

Current Density (mA/m2)

1.6

Voltage (V)

1.4

0.6

1.2

0.5

0.4

0.8

0.3

0.6

0.2

0.4

mass transport limited

0.1

0.2

0
0

~1.4 mWatt/m2

0.7

Power Density (mWatt / m )

0.8

Marine sediment &

seawater from an
estuarine site within
Raritan Bay, New
Jersey,
40 o 27.5 N,
74 o 04.4 W
(3.2% Reduced Carbon)

Concentration (mM)
0

20

40

0
1
2

HS-

3
4

Current Density (mA/m )

Reimers CE, Tender LM, Fertig S, Wang W. Harvesting energy from the marine sediment-water interface. Environmental Science & Technology
2001;35:192-5.

BMFC (Benthic Microbial Fuel Cell)

full system integration:

regulate discharge of BUG at 0.35 V
convert 0.35 V to 6V
recharge capacitor to power RF TX for
real-time data
0.02W average draw (0.48 Wh/day, 175
Wh/yr, 10 alkaline D-cells/yr)
diver assisted deployment
ran flawlessly for 7 months until ice flow
severed mooring
J. Power Sources 2008, 179 (2), 571-575.

BMFC (Benthic Microbial Fuel Cell)

Tuckerton clone library results
80

Anode enrichment of iron-reducing microbes

Percent of clone library

70

current
no current

60
Enrichment in -subgroup from 23% to 75%
45% of the enriched are Desulfuromonas
acetoxidans: (oxidize acetate, reduce insoluble
iron/manganese oxides FeRB)
24% are of the enriched are
Desulfobulbus/Desulfocapsa: S0 H2S + S04-2

50
40
30
20
10
0

Science 295 (5554), 483-485 (2002).

Nature Biotechnology 20 (8): 821-825 (2002).

CFB

Gram+ other
16S ribosomal DNA (rDNA) genes

Bioelectrochemical Systems (BES)

Microbial Electrochemisty/Electro-Microbiology
0.5
65 C

Current (mA)

0.4

Cells

Acetate

Columbic
efficiency
99%

0.3

0.2

pure culture D.
Acetoxidans

AQDS

when no mass
transport
limitations:10 A / m2

Columbic
efficiency
85%

Can grown biofilm by

poising electrode at
positive potential
(0.5 V vs. SHE)

0.1

0
-50

50

100

150

Time (h)

Science 295 (5554), 483-485 (2002).

200

250

300

BMFC (Benthic Microbial Fuel Cell)

O2 + H+

H2O

?
Cathode
Oxic
Water
Anoxic
Sediment

e-

eH+

aerobes
O2 + C6H12O6

C6H12O6

CO2 + H2O
Anode

e-

Geobacter
C6H12O6

CH3CO2-

Clostridium
Nat. Biotechnol. 2002, 20 (8), 821-825.

H+ + CO2

The Question:
G. sulfurreducens

anode
10 m

biofilm

anode

e-

eexternal
circuit

Ac

e-

CO2, H+

media

Bond DR, Strycharz-Glaven SM, Tender LM, & Torres CI (2012) On electron transport through Geobacter biofilms. ChemSusChem 5(6):1099-1105.

Model of Extracellular Electron Transport

Extracellular
Cytochrome
(EC)

cell

pili

Electron Hopping
Richter H, et al. (2009) 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 2:506516.
Strycharz SM, et al. (2011) Application of cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified
anodes of Geobacter sulfurreducens strain DL1 vs. variant strain KN400. Energy Environ. Sci. 4(3):896-913.
Strycharz-Glaven SM & Tender LM (2012) Study of the mechanism of catalytic activity of G. sulfurreducens biofilm anodes during
biofilm growth. ChemSusChem 5(6):1106-1118.

Lovley Lab, UMass

Reguera Lab, MSU

Extracellular
Cytochrome
(EC)

cell

pili

Feliciano, G. T.; da Silva, A. J. R.; Reguera, G.;

Artacho, E., Molecular and Electronic Structure of
the Peptide Subunit of Geobacter sulfurreducens
Conductive Phi from First Principles. J. Phys.
Chem. A 2012, 116 (30), 8023-8030.

Mattick, J. S., Type IV pili and

twitching motility. In Annual Review
of Microbiology. Volume 56, 2002;
Vol. Volume 56, pp 289-314.

Extracellular
Cytochrome
(EC)

Molecular structure of MtrF of S. oneidensis MR-1.

Molecular underpinnings of Fe(III) oxide reduction

by Shewanella oneidensis MR-1, Shi, L. et al.,
Front. Microbiol., 15 February 2012 | doi:
10.3389/fmicb.2012.0005

Model of Extracellular Electron Transport

Extracellular
Cytochrome
(EC)

cell

pili

Nernst Equation

Cyclic Voltammetry
E

eAc/CO2
microbe

e-

eexternal circuit, +
apply potential Electrode

Cyclic Voltammetry
E

Ac/CO2
microbe

external circuit,
apply potential

+
Electrode

anode

biofilm

increasing portion
of cytochromes
that are reduced

redox gradient
diffusive EET

1
fraction of ECs in
oxidized state
vs.
distance form the
electrode surface

Ficks 1st Law:

Dahms-Ruff:

Application of cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified anodes of Geobacter sulfurreducens
strain DL1 vs. variant strain KN400, Sarah M. Strycharz, Anthony P. Malanoski, Rachel M. Snider, Hana Yi, Derek R. Lovley and Leonard M. Tender,
Energy Environ. Sci., 2011, 4, 896-913

mapping vibrational activity to the heme structure by using Density Functional Theory

Oxidation State Independent Peak

Oxidation State Dependent Peaks

z = 0 mm
more oxidized

1315

Raman Intensity, a.u.

1.4

1362

z = 0 mm
more reduced
1371
1401

1.2

1412
1.0
0.8
0.6
0.4

ex situ
~1 mm spot diameter
z = 0, 3, 6, 9 mm

0.2
0.0
1300

1320

1340
1360
-1
Raman shift, cm

1380

1400

1420

Lebedev, N.; Strycharz-Glaven, S. M.; Tender, L. M., Spatially Resolved Confocal Resonant Raman Microscopic Analysis of AnodeGrown Geobacter sulfurreducens Biofilms. ChemPhysChem 2014, 15 (2), 320-327.

Spectroscopic Slicing to Reveal Internal Redox Gradients in ElectricityProducing Biofilms

Angewandte Chemie International Edition

Volume 52, Issue 3, pages 925-928, 26 NOV 2012 DOI: 10.1002/anie.201205440
http://onlinelibrary.wiley.com/doi/10.1002/anie.201205440/full#fig2

in situ

Cyclic Voltammetry
Turnover during biofilm growth

EC mechanism
Nernstian

Nicholson & Shain

Diffusive Behavior
Strycharz SM, et al. (2011) Application of cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified
anodes of Geobacter sulfurreducens strain DL1 vs. variant strain KN400. Energy Environ. Sci. 4(3):896-913.

InterDigitated microelectrode Array (IDA)

45 mm
http://www.ijcambria.com/IDA_Electrode
_Cable_kit.pdf

media
biofilm

eElectrode 1

eElectrode 2
15 mm

eElectrode 1

eElectrode 2

15 mm

Snider RM, Strycharz-Glaven SM, Tsoi SD, Erickson JS, & Tender LM (2012) Long-range electron transport in Geobacter
sulfurreducens biofilms is redox gradient-driven. PNAS109(38):15467-15472.

IDA Gate Experiments

media
e- biofilm
e-

eElectrode 1
Drain
-0.475 V
applied

Electrode 2
Source
-0.575 V
applied

Electrode 1
Drain
-0.475 V
applied

e-

eElectrode 2
Source
-0.575 V
applied

IDA Gate Experiments

gate

drain

PE

source

PE
Ac/CO2
microbe

IDA Gate Experiments

gate

drain

PE

source

PE
Ac/CO2
microbe

ee-

e-

IDA Gate Experiments

gate

drain

PE

source

PE
Ac/CO2
microbe

drain

source

B
C

B
w/o Ac

B
C
drain

source

w/ Ac

Electron Transport Across Fledgling Geobacter sulfurreducens Biofilms

Cells only using UV filter

EPS only using TRITC filter

Cells (stained with DAPI) are false colored red

EPS (stained with ConA-TRITC) are false colored green.
Strycharz-Glaven SM, et al. (2014) Electron Transport through Early Exponential-Phase Anode-Grown Geobacter sulfurreducens
Biofilms. ChemElectroChem 1(11):1957-1965.

Electron Transport Across Fledgling Geobacter sulfurreducens Biofilms

Strycharz-Glaven SM, et al. (2014) Electron Transport through Early Exponential-Phase Anode-Grown Geobacter sulfurreducens
Biofilms. ChemElectroChem 1(11):1957-1965.

Acknowledgments

Sarah Glaven
Jeff Erickson
Rachel Snider
Stanislav Tsoi
Nikolai Lebedev

NRL
ONR