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

From Mud to Electrode Catalysts and Conductive Nanomaterials

December 10, 2014

Electrode-grown Geobacter sulfurreducens biofilm Image: Bond Lab, Univ of Minn

Geobacter metallireducens Image: Lovley Lab, UMass

Iron-Reducing Bacteria (FeRB)

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

Cat

ho

de

An

od

e

Bioelectrochemical Systems (BES) Microbial Electrochemistry/Electro-Microbiology

•Bacteria generate electrons

•Bacteria transfer electrons onto electrodes •Bio-anode can be a single species •Anaerobic

Microbial Bioanodes • Bacteria consume electrons. • Bacteria need carbon – CO2. • Cathode needs a consortium. • Bio-cathode consortia/synergy

Micobial Biocathodes

e-

e- e-

O2, CO2 C2HO

CO2

H2O, C2HO

Demonstrated bio-cathodes: • Bioremediation • Denitrification • Oxygen reduction • CO2 fixation

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

e- Biocathdode MCL

(seawater enriched)

Geobacter

Shewanella

mixed communities

FeRB

Mariprofundus ferrooxydans

mixed communities

FeOB

CBMSE 6.2 Review, June 18, 2003

1 - 10 cm

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

vs. SHE -0.2 V 0.6 V water

sediment

Limmol. Oceanogr. 1969, 14, 547-558

Pt microelectrode

w/insulating sheath

CBMSE 6.2 Review, June 18, 2003

Oxygen: (CH2O)106(NH3)16(HPO4) + 138O2 -181

106CO2 + 16HNO3 + H3PO4 + 122H2O

Manganese: (CH2O)106(NH3)16(HPO4) + 236MNO2 + 472H+ -175

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

Nitrate: (CH2O)106(NH3)16(HPO4) + 84.4HNO3 -156

106CO2 + 44.2N2 + H3PO4 + 148.4H2O + 16NH3

Iron: (CH2O)106(NH3)16(HPO4) + 424Fe2O3 + 484H+ -24

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

Sulfate: (CH2O)106(NH3)16(HPO4) + 53SO42- -21

106CO2 + 16NH3 + H3PO4 + 53S2- + 106H2O

Methanogenesis: (CH2O)106(NH3)16(HPO4) -20

53CO2 + 53CH4 + 16NH3 + H3PO4

DGo (kJ/mole C6H12O6 )

Benthic Redox Gradient

1 -

10 c

m

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

organic matter, oxidants

Anode

Cathode

XH H+ + X-

e-

e- e-

O2 + H+ H2O

H+

Anoxic

Sediment

Oxic

Water

O2 + C6H12O6 CO2 + H2O

aerobes

A fuel cell? Why?

Marine sediment &

seawater from a salt

marsh near

Tuckerton, New

Jersey USA,

39o 30.5’ N, 74o19.6’

W.

(5.5% Reduced

Carbon)

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)

Voltage and power density vs. current density for Identical

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

0

1

2

3

4

De

pth

in

Se

dim

en

t (c

m)

0 2000 4000

Concentration (mM)

HS-

0

1

2

3

4

0 20 40

Concentration (mM)

HS-

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8

Current Density (mA/m2)

Vo

ltag

e (

V)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Po

wer

Den

sit

y (

mW

att

/ m

2)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6 8

Current Density (mA/m2)

Vo

ltag

e (

V)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Po

wer

Den

sit

y (

mW

att

/ m

2)~1.2 mWatt/m2

~1.4 mWatt/m2

•low voltage

•ohmic

•mass transport limited

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.

0

10

20

30

40

50

60

70

80

Tuckerton clone l ibrary results

current

no current

CFB Gram+ other

Per

cen

t of

clo

ne

lib

rary

• 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

Science 295 (5554), 483-485 (2002). Nature Biotechnology 20 (8): 821-825 (2002).

Anode enrichment of iron-reducing microbes

16S ribosomal DNA (rDNA) genes

BMFC (Benthic Microbial Fuel Cell)

0

0.1

0.2

0.3

0.4

0.5

-50 0 50 100 150 200 250 300

Time (h)

Cells Acetate AQDS

65 °CC

urr

ent

(mA

)

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

Columbic efficiency

85%

Columbic efficiency

99%

• pure culture D.

Acetoxidans

• when no mass transport limitations:10 A / m2

• Can grown biofilm by poising electrode at positive potential (0.5 V vs. SHE)

Bioelectrochemical Systems (BES) Microbial Electrochemisty/Electro-Microbiology

Anode

Cathode

C6H12O6 CH3CO2- H+ + CO2

Clostridium

e-

Geobacter

e- e-

O2 + H+ H2O

H+

Anoxic

Sediment

Oxic

Water

?

O2 + C6H12O6 CO2 + H2O

aerobes

BMFC (Benthic Microbial Fuel Cell)

C6H12O6

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

external circuit

Ac

CO2, H+ e-

biofilm

anode

media

0 L z

e-

10 µm

anode e- ?

G. sulfurreducens

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

The Question:

Model of Extracellular Electron Transport

cell pili

Extracellular

Cytochrome

(EC)

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 2:506-

516.

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

cell pili

Extracellular

Cytochrome

(EC)

Mattick, J. S., Type IV pili and

twitching motility. In Annual Review

of Microbiology. Volume 56, 2002;

Vol. Volume 56, pp 289-314.

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.

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

cell pili

Extracellular

Cytochrome

(EC)

Nernst Equation

Ac/CO2

microbe

e-

e-

Electrode

external circuit, apply potential

e-

-

+

E

Cyclic Voltammetry

Ac/CO2

microbe

Electrode

external circuit, apply potential

-

+

E

Cyclic Voltammetry

0 L z

anode biofilm

increasing portion of cytochromes

that are reduced

fraction of ECs in oxidized state

vs. distance form the electrode surface

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

1

•redox gradient

•diffusive EET

Fick’s 1st Law:

Dahms-Ruff:

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Ra

ma

n I

nte

nsity,

a.u

.

1420140013801360134013201300

Raman shift, cm-1

137113621315

14011412

Oxidation State Independent Peak Oxidation State Dependent Peaks

z = 0 mm

more oxidized z = 0 mm

more reduced

• ex situ

• ~1 mm spot diameter

• z = 0, 3, 6, 9 mm

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

Lebedev, N.; Strycharz-Glaven, S. M.; Tender, L. M., Spatially Resolved Confocal Resonant Raman Microscopic Analysis of Anode-

Grown Geobacter sulfurreducens Biofilms. ChemPhysChem 2014, 15 (2), 320-327.

Spectroscopic Slicing to Reveal Internal Redox Gradients in Electricity‐Producing 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

Turnover during biofilm growth

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.

Diffusive Behavior

“EC mechanism”

Cyclic Voltammetry

Nernstian

Nicholson & Shain

45 mm

e- e- e- e-

Electrode 1 Electrode 2 Electrode 1 Electrode 2

media

biofilm

15 mm 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.

InterDigitated microelectrode Array (IDA)

http://www.ijcambria.com/IDA_Electrode

_Cable_kit.pdf

biofilm

media

e- e- e- e- e-

Electrode 1 Drain

-0.475 V applied

Electrode 2 Source

-0.575 V applied

Electrode 1 Drain

-0.475 V applied

Electrode 2 Source

-0.575 V applied

IDA Gate Experiments

Ac/CO2

microbe

PE PE

source drain gate

IDA Gate Experiments

Ac/CO2

microbe

e-

PE

e-

PE

source drain gate

IDA Gate Experiments

e-

Ac/CO2

microbe

PE PE

source drain gate

IDA Gate Experiments

A

B

C

A

B

C

drain source

w/o Ac

A

B

C

w/ Ac

drain source

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.

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.

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

Acknowledgments

• Sarah Glaven

• Jeff Erickson

• Rachel Snider

• Stanislav Tsoi

• Nikolai Lebedev

• NRL

• ONR