Geobacter: The Microbe Electric's Physiology, Ecology, and Practical Applications

Geobacter: The Microbe Electrics
Physiology, Ecology, and Practical

Applications
Derek R. Lovley, Toshiyuki Ueki, Tian Zhang, Nikhil S.
Malvankar, Pravin M. Shrestha, Kelly A. Flanagan, Muktak
Aklujkar, Jessica E. Butler, Ludovic Giloteaux, Amelia-Elena
Rotaru, Dawn E. Holmes, Ashley E. Franks, Roberto Orellana,
Carla Risso and Kelly P. Nevin
Department of Microbiology and Environmental Biotechnology Center, University of

Massachusetts, Amherst, Massachusetts, USA
ABSTRACT
Geobacter species specialize in making electrical contacts with extracellular

electron acceptors and other organisms. This permits Geobacter species to
fill important niches in a diversity of anaerobic environments. Geobacter
species appear to be the primary agents for coupling the oxidation of
organic compounds to the reduction of insoluble Fe(III) and Mn(IV)
oxides in many soils and sediments, a process of global biogeochemical
significance. Some Geobacter species can anaerobically oxidize aromatic
hydrocarbons and play an important role in aromatic hydrocarbon removal
from contaminated aquifers. The ability of Geobacter species to reductively
precipitate uranium and related contaminants has led to the development
of bioremediation strategies for contaminated environments. Geobacter
species produce higher current densities than any other known organism in
microbial fuel cells and are common colonizers of electrodes harvesting
electricity from organic wastes and aquatic sediments. Direct interspecies
electron exchange between Geobacter species and syntrophic partners
ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 59 Copyright # 2011 by Elsevier Ltd.

ISSN: 0065-2911 All rights reserved
DOI: 10.1016/B978-0-12-387661-4.00004-5
2 DEREK R. LOVLEY ET AL.
appears to be an important process in anaerobic wastewater digesters.

Functional and comparative genomic studies have begun to reveal
important aspects of Geobacter physiology and regulation, but much
remains unexplored. Quantifying key gene transcripts and proteins of
subsurface Geobacter communities has proven to be a powerful approach
to diagnose the in situ physiological status of Geobacter species during
groundwater bioremediation. The growth and activity of Geobacter species
in the subsurface and their biogeochemical impact under different
environmental conditions can be predicted with a systems biology
approach in which genome-scale metabolic models are coupled with
appropriate physical/chemical models. The proficiency of Geobacter
species in transferring electrons to insoluble minerals, electrodes, and
possibly other microorganisms can be attributed to their unique microbial
nanowires, pili that conduct electrons along their length with metallic-like
conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter
species do not contribute to this long-range electron transport, but
cytochromes are important for making the terminal electrical connections
with Fe(III) oxides and electrodes and also function as capacitors, storing
charge to permit continued respiration when extracellular electron
acceptors are temporarily unavailable. The high conductivity of Geobacter
pili and biofilms and the ability of biofilms to function as supercapacitors
are novel properties that might contribute to the field of bioelectronics.
The study of Geobacter species has revealed a remarkable number of
microbial physiological properties that had not previously been described
in any microorganism. Further investigation of these environmentally
relevant and physiologically unique organisms is warranted.
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Distribution and Abundance of Geobacter Species . . . . . . . . . . . . . . . . . 6
3. Brief Description of Geobacter Species . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4. Phylogeny and Genomic Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
5. Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5.1. Fe(III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.2. Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.3. Other Extracellular Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . 19
5.4. Other MicroorganismsSyntrophy . . . . . . . . . . . . . . . . . . . . . . . . . 21
6. Electron Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.1. Acetate, Other Fatty Acids, Hydrogen, Electrodes, Humics,
Fe(II), U(IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.2. Aromatic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
7. Extracellular Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
7.1. Microbial Nanowires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
7.2. Cytochromes and Multicopper Proteins . . . . . . . . . . . . . . . . . . . . . . 30
GEOBACTER PHYSIOLOGY AND ECOLOGY 3
7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide . . . . . . . 34

7.4. Model for Extracellular Electron Transfer to Electrodes . . . . . . . . . 37
7.5. Extracellular Electron Transfer in Syntrophy . . . . . . . . . . . . . . . . . . 40
7.6. Model for Extracellular Electron Transfer to Other Extracellular
Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7.7. Capacitor Role of Cytochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
8. Regulation of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8.1. Sigma Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8.2. Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
8.3. Two-Component Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.4. Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.5. Nucleotide-Based Second Messenger . . . . . . . . . . . . . . . . . . . . . . . 50
8.6. Summary Statement on Regulation . . . . . . . . . . . . . . . . . . . . . . . . . 51
9. Environmental Systems Biology of Geobacter . . . . . . . . . . . . . . . . . . . . . 52
9.1. Environmental Transcriptomics and Proteomics . . . . . . . . . . . . . . . 52
9.2. BUGS (Bottom-Up Genome-Scale) Modeling . . . . . . . . . . . . . . . . . 54
10. Biogeochemical Impacts of Geobacter Species . . . . . . . . . . . . . . . . . . . . 56
11. Practical Applications of Geobacter Species . . . . . . . . . . . . . . . . . . . . . . 57
11.1. Bioremediation: Natural Attenuation and Engineered . . . . . . . . . . . 57
11.2. Producing Methane from Organic Wastes and
Hydrocarbon Deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
11.3. Microbial Fuel Cells, Electrosynthesis, and Bioelectronics . . . . . . 61
12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1. INTRODUCTION
Geobacter species represent a rare example of a genus of microorganisms

that are abundant and play an important biogeochemical role in a diversity
of natural environments, yet are easily cultured and can be genetically
manipulated for physiological studies. Although there are other Fe(III)-
reducing microorganisms that have been studied in more detail, it is clear
that Geobacter species are generally the predominant Fe(III)-reducing
microorganisms in many soils and sediments in which Fe(III) reduction is
an important process. Physiological studies with Geobacter species have
revealed a number of novel microbial properties that have an important
impact on the geochemistry of some anaerobic soils and sediments and,
in some instances, have practical applications.
As detailed in subsequent sections, the following microbial processes
were first identified in studies with Geobacter species: (1) oxidation of
organic compounds to carbon dioxide with Fe(III) or Mn(IV) as the elec-
tron acceptor, (2) conservation of energy from organic matter oxidation
coupled to Fe(III) or Mn(IV) reduction, (3) production of extracellular
magnetite from microbial Fe(III) reduction, (4) anaerobic oxidation of an

aromatic hydrocarbon in pure culture, (5) microbial reduction of U(VI),
(6) microbial reduction of Co(III), (7) utilization of humic substances as
an electron acceptor for microbial respiration, (8) oxidation of organic
compounds to carbon dioxide with an electrode serving as an electron
acceptor, (9) conservation of energy from the oxidation of organic com-
pounds coupled to electron transfer to an electrode, (10) the potential
for an electrode to serve as an electron donor to support microbial respira-
tion, (11) use of cytochromes as capacitors to permit respiration in the
absence of exogenous electron acceptors, (12) extracellular electron trans-
fer via microbial nanowires, (13) organic metallic-like long-range conduc-
tion of electrons along a protein filament, (14) production of conductive
biofilms with conductivities comparable to that of synthetic polymers,
and (15) the potential for interaction with syntrophic partners via a direct
electron transfer (Fig. 1).
The reduction of Fe(III), and to a lesser extent Mn(IV), by Geobacter
species can play an important role in carbon cycling in water-saturated
soils and aquatic sediments and further influences the geochemistry of
these environments through the release of dissolved Fe(II) and Mn(II) as
Dominance in Current Metallic-like

Genus Geobacter petroleum- conduction in pili
GS-15 reported established production
contaminated and biofilms
aquifer Microbial
Anaerobic nanowires
Anaerobic
aromatic Electrode-driven
benzene
hydrocarbon Humics Genetic dechlorination
degradation
degradation reduction System G. sulfurreducens
genome
1985 1990 1995 2000 2005 2010 2015
U(VI) Humics as Electrode-

reduction donor driven Interspecies
respiration electron
Role of transfer
Reductive Importance in
Additional novel G. sulfurreducens Motility, dechlorination methanogenic
properties of described chemotaxis,
GS-15 reported aggregates
and pili
Figure 1 Time line of important discoveries associated with Geobacter species.

well as trace metals, metalloids, and phosphate that adsorb onto Fe(III)
and Mn(IV) oxides. In fact, the studies that led to the discovery of the first
Geobacter species were initially designed to better understand the flux of
phosphate from aquatic sediments that contributes to algal blooms.
Geobacter reduction of U(VI) and radionuclides can have an important
influence on the migration of these compounds and is considered to be a
potential tool for mitigating environmental contamination. Geobacter spe-
cies play an important role in degrading a diversity of organic contaminants
in groundwater, both under natural attenuation and engineered bioremedi-
ation strategies. The ability of Geobacter species to exchange electrons
with electrodes has inspired several new strategies for bioenergy and
bioremediation. A recent surprise is the realization that Geobacter
species are important syntrophic microorganisms, forming partnerships
with methanogenic microorganisms, under conditions where they can
significantly contribute to the conversion of organic wastes, or hydrocarbon
deposits, to methane. The production of Geobacter-based materials
with novel electronic properties is a newly emerging field of study.
The number of publications on Geobacter species is relatively small but
continues to grow (Fig. 2) as does awareness of the environmental
relevance of these organisms and their potential practical applications.
The purpose of this review is to provide a broad overview of what has been
learned about Geobacter species since they were discovered 25 years ago.
Due to time and space constraints, not every publication mentioning
Geobacter species could be reviewed.
Geobacter items published
140 4500
Geobacter items cited
4000
120
3500
100
each year
each year
3000
80 2500
60 2000
1500
40
1000
20 500
0 0
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
1995
1997
1999
2001
2003
2005
2007
2009
Figure 2 Publications and citations each year with Geobacter as a topic

according to data from the Thomson Reuters ISI Web of Knowledge.
2. DISTRIBUTION AND ABUNDANCE OF GEOBACTER

SPECIES
The hallmark physiological capability of Geobacter species is their ability

to couple the oxidation of organic compounds to the reduction of Fe
(III), which allows Geobacter species to fill key niches in the anaerobic
microbial food chain of sedimentary environments such as aquatic
sediments, wetlands, rice paddies, and subsurface environments in which
Fe(III) reduction is an important terminal electron-accepting process
(Lovley, 1987, 1991, 1993, 1995, 2000b). For example, molecular analysis
of the metabolically active microorganisms in Fe(III)-reducing rice paddy
soils revealed that Geobacter species accounted for 85% of the
microorganisms consuming acetate, the key intermediate in anaerobic deg-
radation of organic matter (Hori et al., 2010). Other factors such as the
remarkably low maintenance energy requirement of Geobacter species
may also be an important factor in their success in subsurface
environments (Lin et al., 2009).
Molecular analyses, which avoid cultivation bias, have generally found
that Geobacter species are the most abundant Fe(III)-reducing
microorganisms in environments in which Fe(III) reduction is actively tak-
ing place (see, e.g., Anderson et al., 2003; Cummings et al., 2003; Holmes
et al., 2007; Hori et al., 2010; Islam et al., 2004a; Kerkhof et al., 2011;
Rooney-Varga et al., 1999; Rling et al., 2001; Snoeyenbos-West et al.,
2000; Stein et al., 2001; Vrionis et al., 2005). Pure culture isolates have been
recovered from a diversity of environments (Table 1). Further, molecular
(Fig. 3) and/or enrichment studies have detected Geobacter in diverse
environments such as aquifers contaminated with petroleum (Alfreider
and Vogt, 2007; Anderson et al., 1998; Botton et al., 2007; Coates et al.,
1996; Holmes et al., 2007; Prakash et al., 2010; Rooney-Varga et al., 1999;
Salminen et al., 2006; Snoeyenbos-West et al., 2000; Van Stempvoort
et al., 2009; Winderl et al., 2007, 2008); groundwater contaminated with
landfill leachate (Kuntze et al., 2011; Lin et al., 2005, 2007; Rling et al.,
2001; Staats et al., 2011); environments contaminated with organic acids
(Azizian et al., 2010; Stults et al., 2001); contaminated soils and aquatic
sediments (Blothe et al., 2008; Cummings et al., 2000; Haller et al., 2011;
Halm et al., 2009; Manickam et al., 2010); uranium-contaminated subsur-
face sediments amended with organics to promote metal reduction (Akob
et al., 2008; Amos et al., 2007; Anderson et al., 2003; Baldwin et al., 2008;
Brodie et al., 2006; Burkhardt et al., 2010, 2011; Callister et al., 2010;
Cardenas et al., 2008, 2010; Chandler et al., 2010; Chang et al., 2005;
t0005 Table 1 Geobacter species available in pure culture listed in the order in which the species were described.
Genome Electron donors Other Optimal

size and oxidized with Fe Fe forms electron growth
Name Source informationa (III)b reducedc acceptorsa,d temperature Referencese
Geobacter Aquatic 4,011,182 bp Ac, Bz, Bzef, PCIO, Fe Mn(IV), 30 Lovley et al.
metallireducens sediments GC%59.5 BtOH, Buty, Bzo, (III)-Cit Tc(VII)*, (1987,
Aklujkar BzOH, p-Cr, U(VI), 1993),
et al. (2009) EtOH, p-HBz, AQDS, Lovley and
p-HBzo, humics, Phillips
p-HBzOH, IsoB, Nitrate (1988a,b)
IsoV, Ph, Prop,
PrOH, Pyr,
Tol, Val
Geobacter Contaminated 3,814,139 bp Ac, H2 PCIO, Fe Tc(VII)*, 35 Caccavo
sulfurreducens ditch GC%60.9 (III)-Cit, Co(III), et al. (1994),
Strain Fe(III)-P U(VI), Lin et al.
PCA AQDS, S , (2004)
Mth et al. Fum, Mal,
(2003) O2
Strain
KN400
Nagarajan
et al. (2010)
Geobacter Contaminated Ac, EtOH, For, PCIO, Fe Mn(IV), 30 Coates et al.
humireducens wetland H2, Lac (III)-Cit AQDS S , (1998)
(strain JW3) nitrate,
Fum,
Geobacter Deep Ac, EtOH For, PCIO, Fe- Mn(IV), 25 Coates et al.
chapellei subsurface Lac NTA AQDS, (2001)
Fum
Geobacter Aquatic Ac, Buty, EtOH, PCIO, Fe AQDS 30 Coates et al.
grbiciae sediments For, Prop, Pyr (III)-Cit (2001)
(continued)
Table 1 (continued)

Geobacter Contaminated Ac, Buty, Bzo, PCIO, Fe AQDS, 30 Coates et al.

hydrogenophilus aquifer EtOH, For, H2, (III)-Cit Fum (2001)
Prop, Pyr, Suc
Geobacter Freshwater Ac, BtOH, Buty, PCIO Mn(IV), 30 Straub and
bremensis ditch Bzo, EtOH, For, So, Fum, Buchholz-
Fum, H2, Lac, Mal Cleven
Mal, Prop, PrOH, (2001)
Pyr, Succ
Geobacter Freshwater Ac, EtOH, For, PCIO, Mn(IV), 30 Straub and
pelophilus ditch Fum, H2, Mal, Akaganeite So, Fum, Buchholz-
Prop, PrOH, Pyr, Mal Cleven
Succ (2001)
Geobacter Fe(III)- 4,615,150 bp Ac, Bzo, BtOH, Fe(III)-Cit, AQDS, 30 Nevin et al.
bemidjiensis reducing GC%60.3 Buty, EtOH, Fe(III)- Fum, Mal, (2005)
subsurface Aklujkar Fum, H2, IsoB, NTA, Fe Mn(IV)
sediment et al., 2010 Lac, Mal, Prop, (III)-P,
Pyr, Succ, Val PCIO
Geobacter Acetate- Ac, BtOH, EtOH, Fe(III)-Cit, AQDS, 1730 Nevin et al.
psychrophilus impacted For, Lac, Mal, Fe(III)- Electrode, (2005)
aquifer Pyr, Succ NTA, Fe Fum, Mal,
sediment (III)-P, Mn(IV)
PCIO
Geobacter Freshwater Ac, Bze, Bzo, Fe(III)-Cit, PCE, TCE, 35 Sung et al.
lovleyi sediment Buty, Cit, EtOH, PCIO nitrate, (2006)
For, Glu, Lac, Fum, Mal,
MeOH, Prop, So, U(VI),
Succ, Tol, YE, Mn(IV)
Geobacter Sedimentary Ac, Buty, BtOH, Fe(III)-Cit, AQDS, 30 Shelobolina
pickeringii kaolin strata EtOH, MeOH, Fe(III)- Mal, Fum, et al.
Glyc, Lac, Pyr, NTA, Fe Mn(IV), (2007a,b)
Succ, Val (III)-P, S , U(VI)*
PCIO
Geobacter Sedimentary Ac, Buty, BtOH, Fe(III)-Cit, Nitrate, 30 Shelobolina
argillaceus kaolin strata EtOH Glyc, Lac, Fe(III)- Mn(IV), et al.
Pyr, Val NTA, Fe S , U(VI)* (2007b)
(III)-P,
PCIO
Geobacter Subsurface soil Ac, Act, H2 Fe(III)- Mal, Fum, 30 Nevin et al.
thiogenes NTA nitrate, So, (2007),De
TCA Wever et al.
(2000)
Geobacter Uranium- 5,136,364 bp Ac, EtOH, Lac, Fe(III)- AQDS, 32 Shelobolina
uraniireducens contaminated GC%54.3 Pyr NTA, Fe Fum, Mal, et al. (2008)
subsurface (III)-P, Mn(IV),
sediment PCIO, U(VI)*
smectite
Geobacter Tar-oil- Ac, Buty, Bz, Bzo, Fe(III)-Cit, Fum 2532 Kunapuli
toluenoxydans contaminated BzOH, For, m-Cr, PCIO et al. (2010)
sediment Prop, Pyr, Ph,
p-Cr, Tol
Geobacter Heavy metal- 4,304,501 bp Ac, Buty, For Fe(III)-Cit, Fum, Mal, 30 Prakash
daltonii and GC% Bzog, Tolg PCIO So, U(VI) et al. (2010)
hydrocarbon- 53.5%
contaminated
shallow
subsurface
sediment
Geobacter Uranium- 5,277,406 bp Ac, Act, Asp, PCIO, AQDS 2225 Holmes
andersonii contaminated GC%61.2 EtOH, For, Fum, subsurface et al.
strain M18 subsurface Glt, Lac, Mal, Pyr, sediment (2011c)
sediment Succ, YE
Geobacter Uranium- 4,745,806 bp Ac, Act, Ala, Bzo, PCIO, AQDS, 2225 Holmes
remediiphilus contaminated GC%60.5 EtOH, For, Glt, subsurface Mn(IV) et al. (2011c)
strain M21 subsurface Lac, Pyr, Ser, sediment
sediment Succ, Xyl, YE
Geobacter Acetate- Ac, Act, Asp, Bzo PCIO, AQDS, 2225 Holmes
aquiferi impacted Cit, Cys, EtOH, subsurface Mn(IV) et al. (2011c)
strain Ply1 aquifer For, Fum, Lac, sediment
sediment Mal, Pyr, Xyl, YE
(continued)
Table 1 (continued)

Geobacter Acetate- Ac, Act, Bzo, PCIO, 2530 Holmes

plymouthensis impacted Butyr, Fum, Lact, subsurface et al. (2011c)
strain Ply4 aquifer Mal, Pep, Prop, sediment
sediment Pyr, Succ
a
Links for genome information: Geobacter metallireducenshttp://www.ncbi.nlm.nih.gov/genome?
Dbgenome&CmdShowDetailView&TermToSearch18912Geobacter sulfurreducens strain PCAhttp://www.ncbi.nlm.nih.gov/genome?
Dbgenome&CmdShowDetailView&TermToSearch379Geobacter sulfurreducens strain KN400http://www.ncbi.nlm.nih.gov/nuccore/
CP002031.1Geobacter bemidjiensishttp://www.ncbi.nlm.nih.gov/genome?Dbgenome&CmdShowDetailView&TermToSearch22805Geobacter
lovleyihttp://www.ncbi.nlm.nih.gov/genome?Dbgenome&CmdShowDetailView&TermToSearch22459Geobacter uraniireducenshttp://www.
ncbi.nlm.nih.gov/genome?Dbgenome&CmdShowDetailView&TermToSearch20999Geobacter daltoniihttp://www.ncbi.nlm.nih.gov/genome?
Dbgenome&CmdShowDetailView&TermToSearch23754Geobacter andersoniihttp://www.ncbi.nlm.nih.gov/genome?
Dbgenome&CmdShowDetailView&TermToSearch26944Geobacter remediiphilushttp://www.ncbi.nlm.nih.gov/genome?
Dbgenome&CmdShowDetailView&TermToSearch24728.
b
Abbreviations for electron donors and acceptors: acetate (Ac), acetoin (Act), alanine (Ala), anthraquinone-2,6-disulfonic acid (AQDS), aspartic acid
(Asp), benzaldehyde (Bz), benzene (Bze), benzoate(Bzo), benzylalcohol (BzOH), butanol (BtOH), butyrate(Buty), citrate (Cit), p-cresol (p-Cr),
m-cresol (m-Cr), cysteine (Cys), elemental sulfur (So), ethanol (EtOH), formate (For), fumarate (Fum), glucose (Glu), glutamic acid (Glt), glycerol
(Glyc), p-hydroxybenzoate (p-HB), p-hydroxybenzaldehyde (p-HBz), p-hydroxybenzylalcohol (p-HBzOH), hydrogen (H2), isobutyrate (IsoB),
isovalerate (IsoV), lactate(Lac), malate (Mal), methanol (MeOH), manganese oxide (Mn(IV)), peptone (Pep), phenol (Ph), propanol (PrOH),
propionate (Prop), pyruvate (Pyr), serine (Ser), succinate (Succ), tetrachloroethylene (PCE), trichloroethylene (TCE), toluene (Tol), trichloroacetic
acid (TCA), valerate (Val), xylose (Xyl), yeast extract (YE).
c
Fe(III) forms: Poorly crystalline iron oxide (PCIO), ferric citrate (Fe(III)-cit), ferric nitrilotriacetic acid (Fe(III)-NTA), ferric pyrophosphate
(Fe(III)-P)
d,*
Organism has the ability to reduce the metal but not determined whether energy to support growth is conserved from reduction of this metal.
e
Reference in which the capacity to grow via Fe(III) reduction is described, followed by references with other physiological traits.
f
Zhang et al. (2011).
g
Electron donors utilized with fumarate as electron acceptor only.
Figure 3 Neighbor-joining tree showing the phylogenetic relationship within

the genus Geobacter based on 16S rRNA gene sequences. The clone sequences
having > 98% 16S rRNA gene sequence identities were grouped into a single clus-
ter. Cultured representatives (black), including isolates whose genomes are fully
sequenced (red) are shown in the figure. Isolation source and the reference for
both pure culture isolates (blue) and representatives environmental clone
sequences (black) are also shown at the right side of the tree. The sequences
assigned as unpublished in the NCBI and SILVA databases are presented with
their accession number. All sequences (> 1300 bases) were obtained from the
SILVA SSU_106 Ref database (Pruesse et al., 2007) and manually aligned in
ARB program (Ludwig et al., 2004) before the phylogenetic tree construction.
The scale bar represents 10% sequence divergence.
Holmes et al., 2002; Hwang et al., 2009; Istok et al., 2004; Kerkhof et al.,
2011; Michalsen et al., 2007; Mohanty et al., 2008; North et al., 2004; Pea-
cock et al., 2004; Scala et al., 2006; Wan et al., 2005; Wilkins et al., 2007,
2009; Williams et al., 2011; Xu et al., 2010); subsurface environments with
high arsenic concentrations (Hry et al., 2008; Islam et al., 2004a; Lear
et al., 2007; Weldon and MacRae, 2006); environments contaminated with
chlorinated compounds or dechlorinating enrichment cultures (Amos et al.,
2007; Bedard et al., 2007; Imfeld et al., 2010; Kim et al., 2010; Macbeth
et al., 2004; Sorensen et al., 2010; Sung et al., 2006; Yoshida et al., 2005);
wetland and aquatic sediments (Brofft et al., 2002; Cifuentes et al., 2000;
Coates et al., 1998; Costello and Schmidt, 2006; Costello et al., 2009; Martins
et al., 2011; Musat et al., 2010; Roden et al., 2006, 2008; Stein et al., 2001;
Straub et al., 1998); freshwater seeps (Blothe and Roden, 2009; Bruun
et al., 2010; den Camp et al., 2008); acidic springs, peat, or sediments
(Adams et al., 2007; Kusel et al., 2008, 2010; Percent et al., 2008); pristine
aquifers (Flynn et al., 2008; Holmes et al., 2007); rice paddy or other soils
(Cahyani et al., 2008; Conrad et al., 2007; Friedrich et al., 2004; Hansel
et al., 2008; Hiraishi et al., 2005; Hori et al., 2010; Ishii et al., 2009; Noll
et al., 2005; Scheid et al., 2004; Zhu et al., 2009); soil rhizosphere (Fernando
et al., 2008); mangrove sediments (Zhang et al., 2008); a 1700-year-old
wooden spear shaft (Helms et al., 2004); iron-rich snow (Kojima et al.,
2009); clay wall material (Kitajima et al., 2008); dental unit water supply sys-
tems (Singh et al., 2003); methanogenic digesters (Cervantes et al., 2003,
2004; Morita et al., 2011; Riviere et al., 2009; Tsushima et al., 2010; Werner
et al., 2011); and the deep subsurface (Coates et al., 1996, 2001; Kovacik
et al., 2006; Shimizu et al., 2006). In many of these studies, it was concluded
that Geobacter species had an important role in influencing the soil/sedi-
ment/groundwater biogeochemistry and/or promoting bioremediation.
Another environment in which Geobacter species or closely related
Desulfuromonas, Geopsychrobacter, and Pelobacter species are often
abundant is on the surface of electrodes harvesting electricity from organic
matter in wastewater or systems initiated with wastewater inocula
(Aelterman et al., 2008; Borole et al., 2009; Butler et al., 2010a; Call
et al., 2009; Chang et al., 2008; Choo et al., 2006; Cusick et al., 2010; Freguia
et al., 2010; Ishii et al., 2008; Jung and Regan, 2007, 2011; Kiely et al., 2011a,b;
Kim et al., 2007, 2008b; Lee et al., 2003, 2008, 2009; Li et al., 2010; Liu et al.,
2008; Luo et al., 2010; Parameswaran et al., 2010; Shimoyama et al., 2009;
Torres et al., 2009a; Xing et al., 2009), as well as sediments (Bond et al., 2002;
De Schamphelaire et al., 2010; Holmes et al., 2004a,d; Kato et al., 2010; Liu
et al., 2007; Reimers et al., 2006; Tender et al., 2002; White et al., 2009; Williams
et al., 2010).
3. BRIEF DESCRIPTION OF GEOBACTER SPECIES
A significant number of pure culture isolates of Geobacter species are

available (Table 1; Fig. 3). All Geobacter isolates are Gram-negative rods
that are capable of oxidizing acetate with the reduction of Fe(III). Other
commonly conserved features include the ability to reduce Mn(IV),
U(VI), elemental sulfur, and humic substances or the humic substance ana-
log anthraquinone-2,6-disulfonate (AQDS). Many isolates have the ability
to use other small molecular weight organic acids, ethanol, or hydrogen as
an electron donor (Table 1).
The two most heavily studied Geobacter species have been G. meta-
llireducens and G. sulfurreducens. G. metallireducens was the first Geobacter
species recovered in pure culture (Lovley and Phillips, 1988a; Lovley et al.,
1987, 1993a). It was with this isolate that many of the novel physiological
attributes listed in Section 1 were discovered. The recent development of a
genetic system for G. metallireducens (Tremblay et al., 2011a) is likely to
refocus attention on this organism to elucidate the physiology of important
novel properties, such as anaerobic benzene degradation.
Geobacter sulfurreducens was the first Geobacter species for which met-
hods for genetic manipulation were developed (Aklujkar and Lovley, 2010;
Coppi et al., 2001; Kim et al., 2005; Lloyd et al., 2003; Park and Kim, 2011;
Rollefson et al., 2009; Ueki and Lovley, 2010a), and therefore it has served
as the Geobacter of choice for functional genomic studies designed to
understand Geobacter metabolism, gene regulation, and extracellular elec-
tron transfer. It was the first Geobacter species found to use hydrogen as
an electron donor, or to grow with elemental sulfur as an electron accep-
tor. The originally isolated strain was referred to as strain PCA (Caccavo
et al., 1994). A commonly used strain of G. sulfurreducens derived from
strain PCA is frequently referred to as strain DL-1 (Coppi et al., 2001)
because this culture was maintained for many transfers in the laboratory
and may have accumulated a significant number of mutations that were
not present in the originally isolated PCA strain. For example, the DL-1
strain only poorly reduces Fe(III) oxide unless it is adapted for growth
on Fe(III) oxide for long periods of time. The capacity for effective Fe
(III) oxide reduction was recovered via adaptive evolution (Tremblay
et al., 2011b).
Another valuable strain of G. sulfurreducens is strain KN400, which was
recovered in a study designed to adaptively evolve G. sulfurreducens for
growth on electrodes (Yi et al., 2009). Although the KN400 and DL-1
strains have an identical 16S rRNA gene sequence, they have some
important physiological differences. In addition to producing more current

than DL-1 (Yi et al., 2009), KN400 also reduces Fe(III) oxides much faster
(Flannagan et al., 2011). One reason for this may be greater expression of
pili in KN400, which, as discussed below, is thought to be a major conduit
for electron transfer to Fe(III) oxide. Further, strain KN400 is motile,
whereas strain DL-1 is not. This can be attributed to interruption of the
gene for the master regulator for flagella gene expression, FrgM, in DL-1
(Ueki et al., 2011). Motility is important in Fe(III) oxide reduction, as
described below, and flagella could play a role in biofilm formation on
electrodes.
Some Geobacter isolates have been isolated in studies focused on novel
physiological properties such as the ability to use aromatic compounds
(G. toluenoxydans; Kunapuli et al., 2010) or reduction of Fe(III) in clays
(G. pickeringii, G. argillaceus; Shelobolina et al., 2007b). G. lovleyi (Sung
et al., 2006) is the only Geobacter species that has been shown to reduc-
tively dechlorinate the chlorinated solvents tetrachloroethylene (PCE)
and trichloroethylene (TCE) that are common groundwater contaminants
and 16S rRNA gene sequences closely related to the pure culture have
been recovered in dechlorinating enrichment cultures (Daprato et al.,
2007; Dennis et al., 2003; Duhamel and Edwards, 2006, 2007; Duhamel
et al., 2004) as well as subsurface environments contaminated with
chlorinated solvents (Amos et al., 2007; Kim et al., 2010; Macbeth et al.,
2004; Sorensen et al., 2010; Sung et al., 2006). G. (formerly Trichlorobacter)
thiogenes is the only other known dechlorinating Geobacter, reducing
trichloroacetic acid (De Wever et al., 2000; Nevin et al., 2007).
One of the goals of isolating pure cultures of Geobacter species is to
obtain isolates that are representative of the Geobacter species that pre-
dominate in environments of interest. Therefore, the isolates Geobacter
uraniireducens (Shelobolina et al., 2008), Geobacter andersonii (Holmes
et al., 2011c), and Geobacter remediiphilus (Holmes et al., 2011c) are of spe-
cial interest because their 16S rRNA gene sequences match 16S rRNA gene
sequences that were found to predominate during active Fe(III) reduction in
the uranium-contaminated aquifer in Rifle, CO when it is amended with
acetate. These isolates may be particularly useful for elucidating the physiol-
ogy of Geobacter species in such systems.
The isolates from the Rifle, CO site are in a phylogenetic clade, known as
subsurface clade I, which as discussed in the next section, is a phylogenetically
coherent group of Geobacter species that have been found to predominate
in a diversity of aquifers in which Fe(III) reduction is important
(Holmes et al., 2007). Other isolates that are also in the clade include
G. bemidjiensis, G. humireducens, G. bremensis, G. daltonii, and
G. plymouthensis. Some of these isolates were recovered from surface

sediments (Table 1).
Subsurface clade II of the Geobacter species includes some subsurface
isolates such as G. chapellei, which was isolated from a deep subsurface
aquifer in which Fe(III) reduction was important (Lovley et al., 1990).
G. psychrophilus and Geobacter aquiferi were isolated from the same ace-
tate-impacted aquifer from which the subsurface clade I isolate
G. plymouthensis was derived (Table 1).
4. PHYLOGENY AND GENOMIC RESOURCES
Geobacter species are in the family Geobacteraceae, which is within the

domain Bacteria, phylum Proteobacteria, class Deltaproteobacteria, and
order Desulfuromonadales. The order Desulfuromonadales branches phy-
logenetically between the orders Syntrophobacterales and Desulfarculales.
The Geobacteraceae family can be further divided into three distinct
clusters: Geobacter, Desulfuromonas, and Desulfuromusa (Holmes et al.,
2004b). The genera Malonomonas and Geopsychrobacter fall within the
Desulfuromusa cluster, Geothermobacter and Geoalkalibacter fall within
the Desulfuromonas cluster, and Pelobacter species are scattered through-
out all three clusters (Fig. 4).
Comparative genomics suggest that the last common ancestor of the
Geobacteraceae was an acetate-oxidizing, respiratory species capable of extra-
cellular electron transfer, and that specialization for fermentative/syntrophic
growth in Pelobacter species evolved at least twice (Butler et al., 2009).
Pelobacter species have lost numerous genes, including most of the c-type
cytochromes, important in extracellular electron transfer, while gaining
unique genes for fermentative and syntrophic growth (Butler et al., 2009;
Haveman et al., 2006). P. carbinolicus may have lost multiheme c-type cyto-
chrome genes and other genes with multiple closely spaced histidine codons,
including the 14-subunit NADH dehydrogenase complex, due to an autoim-
mune response of its CRISPR locus against the histidyl-tRNA synthetase gene
(Aklujkar and Lovley, 2010). Initial studies suggested that Pelobacter
species could reduce Fe(III) (Lovley et al., 1995); further investigation with
P. carbinolicus revealed that Fe(III) reduction was indirect through a sulfur
shuttle (Haveman et al., 2008). P. carbinolicus can grow as a syntroph (Schink,
1992) but does this via interspecies hydrogen transfer rather than the direct
electron transfer that has been documented with Geobacter species (Summers
and Lovley, 2011). Early on it was proposed that P. propionicus should be
f0020 Figure 4 Maximum-likelihood tree showing the phylogenetic relationship
between the members of the family Geobacteraceae within the class
Deltaproteobacteria using 16S rRNA gene (> 1300 bp). The numbers at the branch
points are tree puzzle support values. Only values greater than 50 are shown. For
further methodological details, see the legend to Fig. 3.
placed in the genus Geobacter (Lonergan et al., 1996), but its significantly dif-
ferent evolutionary trajectory and physiology might warrant a separate genus
designation.
Geobacter species can be grouped (Fig. 3) into three distinct clades:
subsurface clade 1, subsurface clade 2, and the G. metallireducens
clade (Holmes et al., 2007). Molecular studies have shown that most of
the Geobacter species that predominate in Fe(III)-reducing subsurface
environments fall into subsurface clade 1 or subsurface clade 2
(Holmes et al., 2007).
Nine Geobacter genomes and two Pelobacter genomes have been
completely sequenced, including two strains of G. sulfurreducens (Table 1).
Descriptions and comparisons of these genomes are available (Aklujkar
et al., 2009, 2010; Butler et al., 2009, 2010b) as are in silico metabolic
models based on these genomes (Mahadevan and Lovley, 2008;
Mahadevan et al., 2006, 2011; Scheibe et al., 2009; Segura et al., 2008; Sun
et al., 2009, 2010; Yang et al., 2010) and other computational analyses
(Krushkal et al., 2007, 2011; Mahadevan et al., 2006; Qu et al., 2009; Tran
et al., 2008; Yan et al., 2007). Also datasets of numerous genome-scale tran-
scriptional and proteomic analyses that may be a useful resource are avail-
able (Ahrendt et al., 2007; Butler et al., 2007; Conlon et al., 2009; Ding
et al., 2008; Franks et al., 2010b; Holmes et al., 2006, 2009; Khare et al.,
2006; Kim et al., 2008a; Krushkal et al., 2007; Leang et al., 2009; Methe
et al., 2005; Nevin et al., 2009; Nunez et al., 2006; Postier et al., 2008; Qiu
et al., 2010; Strycharz et al., 2011a; Yan et al., 2006).
5. ELECTRON ACCEPTORS
Geobacter species can use a diversity of electron acceptors to support anaer-

obic growth (Table 1), and there is evidence that G. sulfurreducens can grow
via oxygen reduction at low oxygen tensions (Lin et al., 2004). Soluble elec-
tron acceptors that can be reduced intracellularly include nitrate, fumarate,
and chlorinated compounds (Table 1). Biochemical studies have identified
protein fractions with nitrate- and nitrite-reductase activity (Murillo et al.,
1999; Naik et al., 1993; Senko and Stolz, 2001) in G. metallireducens, and
the fumarate reductase of G. sulfurreducens, which has the dual role of act-
ing as succinate dehydrogenase, was identified with a gene deletion
approach (Butler et al., 2006). Separate subsections discussing major extra-
cellular electron acceptors follow.
5.1. Fe(III)
At the circumneutral pH in most environments in which Geobacter thrive, Fe

(III) is highly insoluble. In subsurface sediments in which Geobacter species
were active, poorly crystalline Fe(III) hydroxides and structural Fe(III) of
phyllosilicates were the Fe(III) sources in the clay fraction that were reduced
(Shelobolina et al., 2004). Fe(III) forms that mimic these are the best insoluble
Fe(III) sources for cultivating Geobacter species. Crystalline Fe(III) oxides,
which microorganisms do not appear to significantly reduce in natural
sediments (Phillips et al., 1993), are poor electron acceptors for the cultivation
of Geobacter species. Initial attempts to enrich for acetate-oxidizing, Fe(III)-
reducing microorganisms with crystalline Fe(III) forms only yielded meth-
ane-producing enrichment cultures. It was the adoption of poorly crystalline
Fe(III) oxide, synthesized by neutralizing Fe(III) chloride solutions, that led
to successful enrichment and isolation of G. metallireducens. As outlined in
the references in Table 1, subsequent Geobacter species have been enriched
and isolated on a diversity of electron acceptors. However, Fe(III) that either
comes from the environment of interest (Shelobolina et al., 2007b), or closely
resembles that Fe(III), may be most likely to lead to the isolation of the most
environmentally relevant strains.
Reducing the particle size of Fe(III) oxides may accelerate rates of
Fe(III) reduction (Bosch et al., 2010), but whether Geobacter species can
be conveniently cultivated on nanoscale Fe(III) oxides has not yet been
determined. G. metallireducens could use the insoluble Fe(III) complex
Prussian Blue (Fe4[Fe(CN)6]3), an environmental contaminant, as an elec-
tron acceptor to support growth (Jahn et al., 2006).
Geobacter species can be conveniently grown with soluble chelated,
Fe(III) forms (Lovley, 2000b), but whole genome gene expression (Holmes
et al., 2011b) and proteomic (Ding et al., 2008) studies have suggested that
cells grown with soluble Fe(III) have significantly different physiologies than
cells grown on Fe(III) oxide. Soluble Fe(III) appears to be reduced at the
outer cell surface (Coppi et al., 2007), but as discussed below, the number
of proteins that G. sulfurreducens requires for reducing chelated Fe(III) is
significantly less than for Fe(III) oxide reduction.
5.2. Electrodes
After Fe(III) reduction, electron transfer to electrodes is probably the

most studied form of respiration in Geobacter species (Lovley, 2006a,b,
2008b, 2011a; Lovley and Nevin, 2011). All Geobacter species that have
been evaluated have the capacity for electron transfer to electrodes, which,
as discussed in subsequent sections, may have several practical
applications. Geobacter and closely related Desulfuromonas and Geo-
psychrobacter species were the first microorganisms found to conserve
energy to support growth by coupling the oxidation of organic matter with
electron transfer to electrodes and appear to be most effective
microorganisms in carrying out this form of respiration (Bond et al.,
2002; Holmes et al., 2004d; Ieropoulos et al., 2005; Ren et al., 2007). Geo-
bacter or closely related species are frequently the most abundant
microorganisms that colonize electrodes from mixed species inocula or nat-
ural communities, especially under strict anaerobic conditions (see many
references in Section 2). Frequently, the Geobacter species enriched are
most closely related to G. sulfurreducens, which is consistent with the
finding that G. sulfurreducens strains produce the highest current densities
of any known pure cultures and accomplish this with very high (> 90%)
coulombic efficiencies (Nevin et al., 2008; Yi et al., 2009). This is expected
to give them a competitive advantage in colonizing electrodes.
A wide diversity of conductive materials are appropriate electrode
surfaces for Geobacter species. Solid graphite is the most commonly
employed (Bond and Lovley, 2003), but other graphite/carbon materials
(Adachi et al., 2008; Srikanth et al., 2008; Ieropoulos et al., 2010; Selembo
et al., 2010), carbon cloth (Nevin et al., 2008), gold (Richter et al., 2008),
steel (Dumas et al., 2008a), platinum (Marsili et al., 2008; Yi et al., 2009),
and a diversity of conductive polymers (K.P. Nevin, unpublished data)
are also effective.
5.3. Other Extracellular Electron Acceptors
Mn(IV) oxides are typically much less abundant than Fe(III) oxides in soils
and sediments and are more susceptible to abiotic reduction (Lovley and
Phillips, 1988b). There has been much less focus on Mn(IV) reduction than
Fe(III) in Geobacter studies, but many of the aspects of mechanisms for Fe
(III) oxide reduction described below are likely to apply to Mn(IV) reduction.
Geobacter species can reduce a wide diversity of metal ions. It is possible
to grow Geobacter species on some of these electron acceptors when they
are provided in high concentrations in laboratory cultures, but all are gen-
erally found in low abundance in natural environments and would not sup-
port significant amounts of growth. Therefore, it is unlikely that Geobacter
species have evolved specific mechanisms to conserve energy to support
growth with these metal ions as electron acceptors. It seems more likely
that the ability of Geobacter species to reduce metal ions results from the
generalized ability to move electrons to the outer cell surface and onto
redox-carriers that can nonspecifically transfer electrons to a wide variety
of redox-active species, as described in subsequent sections. The environ-
mental significance of the reduction of these metals is that, in general,
reduction decreases solubility, and hence mobility. This can have impor-
tant geochemical consequences in natural environments, such as aiding in
ore deposit formation and, as described in subsequent sections, can be an
effective bioremediation tool.
For example, the discovery that Geobacter species can reduce soluble
U(VI) to the less soluble U(IV) provided a microbial model for U(VI)
reduction in sedimentary environments where U(VI) reduction had previ-
ously been considered to be an abiotic phenomenon and suggested a strat-
egy for removing uranium from contaminated waters (Lovley et al., 1991).
Some Geobacter species can grow with U(VI) as the sole electron acceptor
(Lovley et al., 1991; Sanford et al., 2007), but even in contaminated
environments, U(VI) availability is much less than that of Fe(III), which
supports most of the Geobacter growth (Finneran et al., 2002a).
G. sulfurreducens can use Co(III)-EDTA as an electron acceptor to
support growth (Caccavo et al., 1994). 60Co(III) chelated with EDTA is
a contaminant of nuclear operations and is highly mobile, whereas the
Co(II) produced from microbial reduction is much less mobile (Gorby
et al., 1998).
G. metallireducens conserved energy to support growth from the reduc-
tion of soluble V(V) to less soluble V(IV) with acetate as the electron
donor (Ortiz-Bernad et al., 2004b). In groundwater in which V(V) was a
co-contaminant with U(VI), stimulating the growth of Geobacter effec-
tively removed V(V). However, abiotic reduction of V(V) with Fe(II) pro-
duced from Fe(III) oxide reduction may have also contributed to V(V)
removal (Ortiz-Bernad et al., 2004a).
In a similar manner, Geobacter species may enzymatically reduce
Tc(VII), but abiotic reduction by Fe(II), produced by Geobacter species
or other organisms, is a more likely reaction in soils and sediments (Lloyd
and Macaskie, 1996; Lloyd et al., 2000). G. metallireducens appeared to
reduce Cr(VI) (Lovley et al., 1993a), but this is another species that
Fe(II) can readily reduce abiotically. Other contaminant radionuclides that
Geobacter species can reduce include Np(V) (Lloyd et al., 2000) and Pu
(IV) (Boukhalfa et al., 2007; Rusin et al., 1994).
Geobacter species can reduce Ag(I) precipitating Ag(0) (Law et al.,
2008; Lovley et al., 1993a) and Hg(II) reduction has also been reported
(Lovley et al., 1993a; Wiatrowski et al., 2006). Although a diversity of other
Fe(III)-reducing microorganisms reduced Au(III), the Geobacter species

tested did not (Kashefi et al., 2001).
Some Geobacter species have the ability to reduce elemental sulfur
(Table 1). Elemental sulfur is expected to be abundant in sediments near
the interface of the zones of Fe(III) reduction and sulfate reduction
because sulfide produced in the sulfate reduction zone and entering a zone
containing Fe(III) will abiotically reduce the Fe(III) with the formation of
elemental sulfur. G. metallireducens (Kaden et al., 2002) and possibly other
Geobacter species may be sensitive to sulfide, therefore the reported
inability of some Geobacter species to grow with elemental sulfur as the
electron acceptor may be related to sulfide sensitivity or inappropriate cul-
ture conditions. As discussed below, the mechanisms for elemental sulfur
reduction in G. sulfurreducens are expected to be rather nonspecific, and
it is expected that all Geobacter species should be capable of transferring
electrons to sulfur in a similar manner.
Humic substances are a heterogeneous class of complex organic com-
pounds that can be the most abundant form of organic matter in some soils
and sediments. The structure of humic substances is poorly understood,
but it is known that they contain quinone moieties that are potential elec-
tron acceptors for Geobacter species and some other microorganisms
(Coates et al., 1998; Jiang and Kappler, 2008; Klapper et al., 2002; Lovley
and Blunt-Harris, 1999; Lovley et al., 1996a, 1998; Roden et al., 2010; Scott
et al., 1998; Wolf et al., 2009). If Fe(III) is also available, the hydroquinone
moieties produced when the quinones are reduced can abiotically reduce
Fe(III) to Fe(II), regenerating the quinone state. In this manner, the humic
substances or humic substance analogs, such as AQDS, function as elec-
tron shuttles between the Geobacter species and the Fe(III). Providing
humic electron shuttles can accelerate the reduction of Fe(III) oxide and
make it feasible for Geobacter species to reduce some Fe(III) forms, such
as crystalline Fe(III) oxides, that are otherwise only poorly reduced
(Lovley et al., 1996a, 1998).
5.4. Other MicroorganismsSyntrophy
In the absence of alternative electron acceptors, Geobacter species can

transfer electrons to syntrophic partners. For example, early studies
demonstrated that most Geobacter species could produce hydrogen from
a variety of organic electron donors (Cord-Ruwisch et al., 1998), suggesting
that these Geobacter species might form syntrophic associations with the
well-established principle of interspecies hydrogen transfer (McInerney
et al., 2009; Stams and Plugge, 2009) in which one microorganism disposes
of electrons via the production of hydrogen gas and the partner organism
consumes the hydrogen with electron transfer to an electron acceptor the
other partner cannot utilize.
Acetate-oxidizing cocultures could be established with G. sulfurreducens
and either Wolinella succinogenes or Desulfovibrio vulgaris the first time
that rapid anaerobic syntrophic oxidation of acetate at moderate
temperatures was demonstrated (Cord-Ruwisch et al., 1998). However,
growth yields of G. sulfurreducens in the coculture with W. succinogenes
were higher than expected if G. sulfurreducens was disposing of electrons
via hydrogen production. This initially led to the suggestion that a soluble
c-type cytochrome released in the medium served as an electron shuttle
between the two species (Cord-Ruwisch et al., 1998), followed by the sug-
gestion that cysteine added to the medium was the electron shuttle (Kaden
et al., 2002). A third alternative, currently under investigation, is direct
interspecies electron transfer as described below.
Cell suspensions of G. metallireducens and W. succinogenes oxidized
toluene with the reduction of fumarate (Meckenstock, 1999). The electron
carrier between the two organisms was not investigated, but the poor
capacity for G. metallireducens to produce hydrogen would suggest
that an alternative to interspecies hydrogen transfer might have been
necessary.
The ability of Geobacter species to form syntrophic interactions was fur-
ther investigated in cocultures of G. metallireducens and G. sulfurreducens
(Summers et al., 2010). Multiple lines of evidence suggested that as the
coculture adapted for rapid syntrophic growth, electrons were directly
exchanged between the two species via electrically conductive connections
that will be described later. As detailed later, there is increasing evidence
that direct interspecies electron transfer may be an important feature of
Geobacter in anaerobic environments (Lovley, 2011a,c, Morita et al., 2011).
Another possibility in natural environments is that environmental
components may aid in interspecies electron transfer. For example, in the
reduced state, humic substances and other organics that have quinone/
hydroquinone moieties can serve as electron donors to support anaerobic
respiration (Lovley et al., 1999). G. metallireducens was able to transfer
electrons derived from acetate oxidation much more rapidly to
W. succinogenes in the presence of the humics analog AQDS, which served
as an electron acceptor for acetate oxidation by G. metallireducens, and the
reduced AQDS served as an electron donor for W. succinogenes (Lovley
et al., 1999).
6. ELECTRON DONORS
6.1. Acetate, Other Fatty Acids, Hydrogen, Electrodes,

Humics, Fe(II), U(IV)
The universal ability of all Geobacter species to oxidize acetate with Fe

(III) serving as the sole electron-acceptor points to their key ecological/
biogeochemical role in soils and sediments. Acetate is the key extracellular
intermediate in the anaerobic degradation of organic matter (Lovley and
Chapelle, 1995). Although there are some Fe(III)-reducing
microorganisms that can completely oxidize fermentable organic com-
pounds, such as sugars and amino acids (Lovley et al., 2004), they do not
appear to be competitive with fermentative microorganisms. For example,
in sediments in which Fe(III) reduction was the predominant terminal elec-
tron-accepting process, glucose was metabolized to acetate and other
minor fermentation acids (Lovley and Phillips, 1989). Therefore, minerali-
zation of organic matter can only take place if there are Fe(III) reducers
capable of coupling the oxidation of acetate to the reduction of Fe(III).
Further, acetate is a common additive to stimulate the activity of Geo-
bacter species for in situ bioremediation of uranium-contaminated ground-
water. Therefore, understanding acetate metabolism is key for
understanding the ecology of Geobacter species.
Acetate is oxidized via the TCA cycle in Geobacter species (Champine
and Goodwin, 1991; Champine et al., 2000; Mikoulinskaia et al., 1999).
As recently reviewed in detail (Mahadevan et al., 2011), studies on acetate
transporters (Risso et al., 2008a) as well as iterative genome-scale meta-
bolic modeling and experimental studies (Mahadevan et al., 2006; Risso
et al., 2008b; Segura et al., 2008; Tang et al., 2007) have identified addi-
tional features of the pathways for acetate metabolism that will not be
repeated here.
Even though the Geobacter species whose genomes have been
sequenced were isolated from geographically diverse environments, there
is high conservation of acetate metabolism genes including the genes
encoding acetate transporters and the eight enzymes of acetate oxidation
via the TCA cycle (citrate synthase, aconitase, isocitrate dehydrogenase,
2-oxoglutarate:ferredoxin oxidoreductase, succinyl:acetate CoA-transferase,
succinate dehydrogenase/fumarate reductase, fumarase, and malate dehy-
drogenase) (Butler et al., 2010b). All the genomes encode a 14-subunit
NADH dehydrogenase complex, except G. lovleyi encodes a 12-subunit
NADH dehydrogenase complex. All except G. metallireducens encode the
NAD-dependent ferredoxin:NADP oxidoreductase (Nfn) complex, and all

encode a putative NADPH:menaquinone oxidoreductase (Sfr) complex
(Coppi et al., 2007). The genes that encode ATP synthase are divided into
two conserved operons in every Geobacter genome (Butler et al., 2010b).
In addition to acetate, other short-chain fatty acids, alcohols, and hydro-
gen serve as electron donors for some Geobacter species (Table 1). In
G. sulfurreducens, the enzyme required for hydrogen oxidation has been
identified as a four-subunit NiFe hydrogenase (Coppi et al., 2004). How-
ever, this enzyme is only conserved in G. sulfurreducens,
G. uraniireducens, G. bemidjiensis, G. andersonii, G. remediiphilus,
and G. lovleyi (Butler et al., 2010b). In addition to the respiratory hydrog-
enase, there are multiple other cytoplasmic and membrane-bound hydro-
genases found in Geobacter species, all quite distinct from the well-
studied hydrogenases in related Desulfovibrio species (Coppi, 2005). There
is a hydrogenase specific to Geobacter that predominate in the subsurface
(Butler et al., 2010b).
Genome analysis suggests that G. metallireducens metabolizes propio-
nate via the 2-methylcitrate pathway (Aklujkar et al., 2009; Bond et al.,
2005), whereas G. bemidjiensis is predicted to metabolize propionate via
the methylmalonyl-CoA pathway (Aklujkar et al., 2010). Activation of
butyrate and valerate with a kinase and a transferase to form butyryl-
CoA and valeryl-CoA, respectively, has been predicted from genome
sequences (Aklujkar et al., 2009, 2010), but the expression of butyrate
kinase did not increase in G. metallireducens fed butyrate (Peters et al.,
2007a). G. bemidjiensis also possesses a long-chain fatty acyl-CoA dehy-
drogenase, fadE, which is absent from the genome of G. metallireducens,
suggesting that G. bemidjiensis has the potential for long-chain fatty acid
metabolism. Long-chain fatty acid degradation was documented in the
closely related Desulfuromonas palmitatis (Coates et al., 1995).
G. metallireducens and G. sulfurreducens could reduce nitrate and fuma-
rate, respectively, with the reduced humic substance analog AQDS
(Lovley et al., 1998). G. metallireducens can oxidize Fe(II) and U(IV) with
nitrate as an electron acceptor (Finneran et al., 2002b; Weber et al., 2006).
However, the capacity to conserve energy to support growth with these
electron donors has not been demonstrated.
Electrodes poised at a low potential can serve as an electron donor for
Geobacter species. Electrode-dependent reduction of nitrate (Gregory
and Lovley, 2005), fumarate (Gregory et al., 2004), U(VI) (Gregory and
Lovley, 2005), and chlorinated compounds (Strycharz et al., 2008) has been
documented, as has the reduction of protons to produce hydrogen gas
(Geelhoed and Stams, 2011). Initial studies have suggested that the route
for electron transfer from electrodes to Geobacter species may be different

from electron flow in the opposite direction for current production (Dumas
et al., 2008b; Strycharz et al., 2011a).
6.2. Aromatic Compounds
As detailed later, Geobacter species are often abundant within zones of

petroleum-contaminated aquifers in which aromatic hydrocarbons are
being removed, and it has been suggested that they can play an important
role in converting hydrocarbons in systems designed for recovery of hydro-
carbon deposits as methane. G. metallireducens was the first Geobacter
species found to degrade aromatic compounds and the first microorganism
of any kind in pure culture found to degrade an aromatic hydrocarbon
(Lovley and Lonergan, 1990; Lovley et al., 1989). Of particular interest is
the ability of G. metallireducens to anaerobically degrade benzene (Zhang
et al., 2011). Other than the hyperthermophile Ferroglobus placidus
(Holmes et al., 2011d), G. metallireducens is the only organism in pure cul-
ture that has been reported to be capable of degrading benzene through
defined anaerobic pathways.
A number of other Geobacter species, isolated from a diversity of con-
taminated environments, are capable of degrading aromatic compounds
(Table 1). Geopsychrobacter electrodiphilus, which is closely related to
Geobacter species, also metabolizes aromatic compounds (Holmes et al.,
2004d). All Geobacter species that are capable of degrading aromatic com-
pounds degrade benzoate. Only four (G. metallireducens, G. grbiciae,
G. toluenoxydans, and G. daltonii) are capable of metabolizing toluene.
The mechanisms for the degradation of aromatic compounds in Geo-
bacter species have primarily been investigated in G. metallireducens. Ben-
zoyl-CoA is a central intermediate in the metabolism of all monoaromatic
substrates (Fig. 5). Benzoyl-CoA reductase catalyzes the dearomatization
of benzoyl-CoA to cyclohexan-1,5-diene-1-carboxyl-CoA via a Birch-type
reduction (Boll, 2005b; Boll and Fuchs, 1995; Boll et al., 2000; Kung
et al., 2009; Peters et al., 2007b). G. metallireducens has a class II ben-
zoyl-CoA reductase, which until recently (Holmes et al., 2011d) was
thought to be found in all anaerobes degrading benzoate (Lffler et al.,
2011). Whereas class I benzoyl-CoA reductases couple the hydrolysis of
two ATP with the transfer of two electrons from reduced ferredoxin to
benzoyl-CoA (Boll, 2005a), the class II enzymes, which were initially dis-
covered in G. metallireducens (Kung et al., 2009), do not have an ATP
requirement (Lffler et al., 2011). In order to overcome the high redox
Figure 5 Aromatic hydrocarbon oxidation pathways in Geobacter species.

Enzymes of the aromatics degradation pathways indicated: benzylsuccinate synthase
(BssABCD), benzylsuccinate CoA-transferase (BbsEF), benzylsuccinyl-CoA dehy-
drogenase (BbsG), phenylitaconyl-CoA hydratase (BbsH), 2-[hydroxy(phenyl)
methyl]-succinyl-CoA dehydrogenase (BbsCD), benzoylsuccinyl-CoA thiolase
(BbsAB), alcohol dehydrogenase (adh), 4-hydroxybenzaldehyde dehydrogenase
(PcmO), benzoate-CoA ligase (BamY), phenylphosphate synthase (PpsABC),
phenylphosphate carboxylase (PpcBDthese are enzymes distinct from the PpcB
and PpcD cytochromes), p-cresol methylhydroxylase (PcmIJ), 4-hydroxybenzoyl-CoA
reductase (PcmRST), benzoyl-CoA reductase (BAmBCDEFGI), cyclohexadienoyl-
CoA hydratase (BamR), hydroxyenoyl-CoA dehydrogenase (BamQ), and oxoenoyl-
CoA hydrolase (BamA).
barrier associated with ring reduction, class II benzoyl-CoA reductases

may be driven by either membrane potential or electron bifurcation (Kung
et al., 2010).
A cluster of eight benzoate-induced genes (Bam BCDEFGHI) is
thought to code for the class II benzoyl-CoA reductase (Kung et al.,
2009). BamBC subunits are sufficient to catalyze the reductive
dearomatization of benzoyl-CoA in vitro (Kung et al., 2010). BamB
contains the tungstopterin active site and an FeS cluster, whereas BamC
contains three more FeS clusters (Kung et al., 2009). BamDEFGHI are
thought to participate in an uncharacterized electron activation process.
Proteome analysis revealed that BamFG was found exclusively in the
membrane fraction (Heintz et al., 2009). Therefore, it was proposed
that the benzoyl-CoA reductase complex is membrane associated. Activity
of class II benzoyl-CoA reductase was also detected in extracts of
G. bemidjiensis and several other obligate anaerobes (Lffler et al., 2011).
Additional reactions (Fig. 5) complete the conversion of benzoyl-CoA to 3-

hydroxypimelyl-CoA (Wischgoll et al., 2005), which is then oxidized to ace-
tyl-CoA and CO2 via beta-oxidation. This process includes the oxidative
decarboxylation of glutaryl-CoA to crotonyl-CoA by the decarboxylating
glutaryl-CoA dehydrogenase of G. metallireducens (Wischgoll et al., 2009).
The toluene degradation pathway of G. metallireducens (Fig. 5) appears
to be similar to the one found in the denitrifier Aromatoleum aromaticum
(Butler et al., 2007; Rabus, 2005; Rabus et al., 2005). The benzylsuccinate
synthase adds toluene as a radical to fumarate to form benzylsuccinate.
Benzylsuccinate is then activated to benzylsuccinyl-CoA, which is
metabolized to benzoyl-CoA (Rabus, 2005; Rabus et al., 2005). Proteomic
and functional genomics studies have suggested that the p-cresol degrada-
tion pathway in G. metallireducens is similar to the one found in Pseudo-
monas putida (Butler et al., 2007; Cronin et al., 1999; Peters et al., 2007a).
PcmO, a 4-hydroxybenzaldehyde dehydrogenase, could be responsible
for the conversion of 4-hydoxybenzaldehyde to 4-hydroxybenzoate in the
p-cresol pathway and for the conversion of benzaldehyde to benzoate in
the benzyl alcohol pathway (Butler et al., 2007). G. metallireducens
expressed more PcmO when grown on p-cresol compared to acetate or
benzoate (Peters et al., 2007a). No experimental evidence suggests that
PcmO is involved in benzyl alcohol oxidation.
Phenol degradation in Geobacteraceae is believed to be similar to that in the
facultative anaerobic denitrifier Thaurea aromatica, which was initially used to
describe the phenol pathway (Butler et al., 2007). Genes coding for
phenylphosphate synthase and carboxylase were upregulated when
G. metallireducens was grown on phenol compared to acetate and benzoate
(Schleinitz et al., 2009). Out of four phenylphosphate carboxylase subunits of
T. aromatica, only homolog genes coding for two subunits were found in the
G. metallireducens and Geobacter daltonii genomes. Despite the absence of
the other genes, phenylphosphate carboxylase activity was still detected
(Schleinitz et al., 2009). Further studies are necessary to understand the bio-
chemistry of phenylphosphate carboxylation in Geobacteraceae. It was
suggested that the benzoate-CoA ligase might also catalyze the conversion
of 4-hydroxybenzoate to 4-hydroxybenzoyl-CoA (Butler et al., 2007). How-
ever, biochemical studies demonstrated that the 4-hydroxybenzoate-CoA
ligase activity in G. metallireducens is found in another enzyme (Peters et al.,
2007a; Wischgoll et al., 2005).
Most of the aromatic degradation genes are found on a 300-kb genomic
island in G. metallireducens (Butler et al., 2007). This region contains 244
genes, which code for, among other things, the benzoyl-CoA reductase,
the benzoate-CoA ligase, phenol and p-cresol degradation pathways, the
cyclohexadienoyl-CoA hydratase, the hydroxyenoyl-CoA dehydrogenase,

the oxoenoyl-CoA hydrolase, and fatty acid oxidation enzymes (Butler
et al., 2007). Identical transposons and repetitive sequences found in this
region indicate that possible horizontal transfer events might be responsi-
ble for the acquisition of those genes (Butler et al., 2007). Most of those
244 genes have no ortholog in Geobacteraceae species that are unable to
degrade aromatic compounds (Butler et al., 2007). Genes coding for the
toluene degradation pathway are found on two operons located in another
genomic island of 167 kb (Butler et al., 2007).
Little is known about the regulatory network associated with aromatic
degradation in Geobacteraceae. Recently a G. metallireducens two-
component system formed by the histidine kinase BamV and the response
regulator BamW was found to regulate the transcription of bamY, the gene
coding for the benzoate-CoA ligase (Jurez et al., 2010). BamVW caused
an increase in the transcription of bamY after exposure to benzoate,
4-hydroxybenzoate, or p-cresol. A transcriptional regulator, which in the
presence of acetate represses the expression of bamA, and possibly other
genes whose expression is induced during growth on benzoate, has been
identified in G. bemidjiensis (Ueki, 2011). Genes for phenol or p-cresol deg-
radation are also transcribed when benzoate is the sole electron source
(Peters et al., 2007a; Schleinitz et al., 2009). However, accumulation of the
corresponding proteins was observed only during growth on phenol or
p-cresol, but not with benzoate (Peters et al., 2007a; Schleinitz et al., 2009).
Therefore, the existence of posttranscriptional regulation mechanism(s)
was proposed to explain these observations.
Many aspects of aromatic degradation pathways are still unknown
and need to be characterized in order to further comprehend the role of
Geobacter species in contaminated environments. These studies should be
accelerated by the recent development of methods for genetic manipulation
of G. metallireducens.
7. EXTRACELLULAR ELECTRON TRANSFER
Effective extracellular electron transfer is one of the hallmark physiologi-

cal features of Geobacter species. The capacity to exchange electrons with
its extracellular environment defines the unique ecological niche of
Geobacter species and is an important feature of the many practical
applications of this genus. Extracellular electron transfer in Geobacter
species is accomplished through unique mechanisms that have yet to be

described in any other organism.
7.1. Microbial Nanowires
One of the most surprising discoveries in the study of extracellular electron

transfer in Geobacter species has been the finding that G. sulfurreducens,
and presumably other Geobacter species, produces pili that are electrically
conductive (Malvankar et al., 2011b; Reguera et al., 2005). Initial
indications that pili were important in extracellular electron transfer came
from the observation that G. metallireducens expressed pili when grown on
Fe(III) or Mn(IV) oxides, but not when grown with soluble, chelated
Fe(III) as the electron acceptor (Childers et al., 2002). Studies on pili in
G. sulfurreducens have demonstrated that this organism can produce
pilin-like filaments from several different proteins, but the most abundant
filaments are those comprising PilA (Klimes et al., 2010).
Deletion of the gene for PilA, the structural pilin protein, inhibited Fe(III)
oxide reduction (Reguera et al., 2005). Conducting atomic force microscopy
demonstrated that the pili were conductive across their diameter (Reguera
et al., 2005). The atomic force microscopy revealed that there were other
proteins associated with the pili, but they acted as insulators. Therefore, it
was proposed that a method for electron transfer to Fe(III) oxide was long-
range electron transport along the pilin filaments. Further, although electron
hopping between cytochromes is the accepted method for biological electron
transfer over distance, it was suggested that cytochromes did not mediate
the electron transport along the pili (Reguera et al., 2005). This concept was
seriously questioned (Shi et al., 2007) because there was no known mechanism
for electron transfer along protein filaments.
However, subsequent studies have provided a mechanism. The pili of
G. sulfurreducens possess metallic-like conductivity comparable to syn-
thetic conducting polymers, such as the organic metal polyaniline
(Malvankar et al., 2011b). When pilin preparations were spotted on a
two-electrode system, they formed a network that conducted electrons
between the two electrodes. Preparations from a pilA deletion mutant
had conductivities comparable to the buffer control. Treating the pilin
preparation to denature any cytochromes that might have remained
associated with the pili had no impact on conductivity. Upon cooling from
room temperature, the pilin conductivity increased exponentially, a hall-
mark of quasi-one-dimensional organic metals. The temperature response
would not have been observed if electron hopping between cytochromes
was responsible for the electron transfer. Studies on the impact of pH

changes on conductivity and X-ray diffraction analysis of purified pilin pre-
parations suggested that pp interchain stacking between aromatic
moieties of pilin amino acids may confer the metallic-like conductivity.
This hypothesis is currently under investigation.
The possibility of electron transport along a protein filament without the
involvement of cytochromes is a paradigm shift in biology. The metallic-like
mechanism for electron transport along the pili of G. sulfurreducens under
in vivo conditions is fundamentally different than the conductivity proposed
for filaments of other microorganism such as Shewanella oneidensis,
which was only demonstrated in fixed preparations and was reported to
be dependent on the presence of cytochromes (Gorby et al., 2006).
7.2. Cytochromes and Multicopper Proteins
One of the most striking features of Geobacter species is their abundant

c-type cytochromes and the large diversity of cytochromes encoded in Geo-
bacter genomes (Butler et al., 2010b; Ding et al., 2006; Mth et al., 2003).
With the exception of G. lovleyi, Geobacter species possess ca. 100 c-type
cytochrome genes per genome (Butler et al., 2010b). There are nine famil-
ies of well-conserved c-type cytochromes, four of which are encoded
together and may constitute a quinone:ferricytochrome c oxidoreductase.
However, most of the cytochromes are poorly conserved among the Geo-
bacter species and some cytochrome families have only been found in a sin-
gle species of Geobacter (Butler et al., 2010b). This, coupled with the fact
that the function of c-type cytochromes has only been significantly studied
in G. sulfurreducens, makes it difficult to make broad generalizations about
cytochrome function in Geobacter species.
One family of c-type cytochromes that is well conserved is the PpcA
family of triheme periplasmic cytochromes. These are among the most
abundant c-type cytochromes in Geobacter species and were first studied
biochemically in the closely related Desulfuromonas acetoxidans (Banci
et al., 1996; Bruschi et al., 1997; Czjzek et al., 2001) and G. metallireducens
(Afkar and Fukumori, 1999; Champine et al., 2000) and then with more
detailed functional studies in G. sulfurreducens.
PpcA purified from G. sulfurreducens contained the expected three
hemes with a molecular weight of 9.6 kDa and a midpoint potential of
169.5 mV (Lloyd et al., 2003). Although PpcA is related to the earlier
studied cytochrome in D. acetoxidans, its redox properties are distinct
(Pessanha et al., 2006). Purified PpcA reduced Fe(III) and other metals,
but its periplasmic location makes direct reduction of Fe(III) unlikely

(Lloyd et al., 2003). The heme groups of PpcA are oriented in parallel or
perpendicular to each other (Morgado et al., 2010b), an arrangement
expected to facilitate rapid electron transfer within and between proteins
(Mowat and Chapman, 2005). Deletion of ppcA did not impact fumarate
reduction but did impact reduction of the extracellular electron-acceptors
Fe(III), AQDS, and U(VI) with acetate as the electron donor. However,
with hydrogen as the electron donor, reduction of extracellular electron
acceptors in the mutant and wild type were comparable.
There are four homologs of PpcA in G. sulfurreducens, designated
PpcB-PpcE. The function of these homologs appears to be different. Sur-
prisingly, deletions of ppcB, ppcC, or ppcE increased rates of Fe(III)
reduction (Shelobolina et al., 2007a). Whereas PpcA appears to be
expressed constitutively, PpcD expression is enhanced during growth on
Fe(III) oxide (Ding et al., 2008). Only PpcB is downregulated in cultures
grown on soluble iron (Ding et al., 2008). Structural and thermodynamic
characterizations such as the organization, redox potential, and oxidation
of hemes further suggest different functions of the homologues (Morgado
et al., 2008, 2010a; Pokkuluri et al., 2010). Only PpcA and PpcD can couple
e/H translocation across the inner membrane (Morgado et al., 2010a),
and only PpcC displays polymerization and redox-dependent conforma-
tional changes (Morgado et al., 2007). Much more research into the func-
tion of these periplasmic cytochromes and their interaction with inner
and outer membrane components is required.
Early studies on G. sulfurreducens found significant Fe(III) reductase
activity in membrane fractions, which involved cytochromes (Gaspard et al.,
1998; Magnuson et al., 2000). One of these cytochromes was purified
(Magnuson et al., 2001) and was most likely the subsequently described
OmcB (Leang et al., 2003). This cytochrome has a molecular weight of
89 kDa, 12 hemes, and gross midpoint potential of 190 mV with some
hemes appearing to have much more negative potentials (Magnuson et al.,
2001). The purified protein was capable of reducing Fe(III) oxide and che-
lated Fe(III). OmcB is embedded in the outer membrane, with a portion of
the molecule exposed to the outer surface (Qian et al., 2007). Deleting the
gene for OmcB inhibited reduction of Fe(III) citrate and Fe(III) oxide
(Leang et al., 2003). Deletion mutants adapted to growth on Fe(III) citrate,
but not Fe(III) oxide (Leang et al., 2005). The presence of multiple RpoS-
dependent promoters upstream of upregulated cytochromes in the Fe(III)
citrate-adapted mutant suggests that an activated RpoS response permitted
G. sulfurreducens to compensate for the loss of OmcB (Krushkal et al., 2009).
OmcB is encoded downstream from another cytochrome, designated

Orf2, in an operon with a third protein of unknown function (Leang and
Lovley, 2005). This operon has been duplicated, and the other copy is imme-
diately downstream. In all the other Geobacter species genomes, there is at
least one operon with similarity to this one, and in several, there are tandem
repeats of the operon as well (Butler et al., 2010b). While the Orf2 cyto-
chrome is well conserved across all species, the sequence of the gene in the
omcB position in the operon varies substantially. In all cases, this gene
encodes a multiheme cytochrome, but some of the sequences are very diver-
gent and cannot be called omcB homologs. Thus, while the operon is con-
served and even duplicated, and the sequence of the Orf2 cytochromes is
well conserved, there may be less pressure for the large outer membrane
cytochrome to maintain a specific sequence.
Whereas OmcB is embedded in the outer membrane, several of the G.
sulfurreducens c-type cytochromes are fully exposed on the outer cell surface.
OmcS is a six-heme c-type cytochrome with a molecular weight of 47 kDa
(Qian et al., 2011). Its midpoint redox potential is 212 mV, more negative
than that of the periplasmic c-type cytochromes. However, the available evi-
dence suggests that individual hemes span a wide range of potentials. The gene
for OmcS is the most upregulated gene during growth on Fe(III) oxide versus
growth on Fe(III) citrate (Holmes et al., 2011b) and this is reflected in the pro-
teome (Ding et al., 2008) and in initial studies that detected omcS transcripts in
cells grown on Fe(III) oxide, but not Fe(III) citrate (Mehta et al., 2005). It is
also highly expressed under some conditions during growth on electrodes
(Holmes et al., 2006) and in cocultures of G. sulfurreducens and G. meta-
llireducens (Summers et al., 2010). Purified OmcS reduced a diversity of poten-
tial extracellular electron acceptors for G. sulfurreducens, including Fe(III)
oxide, U(VI), and humics, and also bound Fe(III) oxide (Qian et al., 2011).
OmcS is specifically associated with the pili of G. sulfurreducens (Leang
et al., 2010) and is required for growth on Fe(III) oxide, but not Fe(III) citrate
(Mehta et al., 2005).
OmcE is another c-type cytochrome found on the outer cell surface, but
its specific localization has yet to be pinpointed. It also has not been
purified but is predicted to have a molecular weight of 32 kDa and four
hemes (Mehta et al., 2005). Expression patterns of OmcE (Ding et al.,
2008; Holmes et al., 2006; Kim et al., 2008a; Nevin et al., 2009), as well as
gene deletions studies (Mehta et al., 2005), suggest that OmcE plays a role
in extracellular electron transfer in wild-type cells, but cells can adapt to
the loss of OmcE.
In contrast to OmcE and OmcS, OmcZ is not required for the reduction
of insoluble Fe(III). However, of all G. sulfurreducens cytochromes studied
to date, only OmcZ is absolutely necessary for high-density current pro-

duction (Nevin et al., 2009). In its mature extracellular form, OmcZ has a
molecular weight of 30 kDa, with eight hemes, including an unusual
CX14CH motif (Inoue et al., 2010). Its midpoint potential is 220 mV,
but as with other multiheme cytochromes individual hemes cover a wide
range of potentials. The purified protein can reduce a range of typical sol-
uble extracellular electron acceptors, and Mn(IV) oxides, but only poorly
reduced Fe(III) oxide. This corresponds with increased expression of
OmcZ during growth on Mn(IV) oxide, but not Fe(III) oxide, versus
growth on Fe(III) citrate (Holmes et al., 2011b). The poor solubility of
OmcZ in water might help maintain it within the extracellular matrix
(Inoue et al., 2010). OmcZ is specifically localized at the biofilmanode
interface in high-current density biofilms (Inoue et al., 2011). It does not
associate with filaments and its expression patterns suggest that its natural
function may be to promote the reduction of extracellular soluble electron
acceptors.
The cytochrome encoded by gene GSU1334 is homologous to OmcZ
and a deletion mutant exhibited defects in Fe(III) oxide and U(VI) reduc-
tion (Shelobolina et al., 2007a). However, caution in interpreting such
phenotypes is warranted without additional study. For example, deleting
genes for several cytochromes predicted to be on the outer surface
inhibited Fe(III) reduction, but this could be attributed to the lack of
proper expression or localization of OmcB or other outer-surface
cytochromes in these mutants (Kim et al., 2005, 2006, 2008a). In a similar
manner, deletion of the gene for MacA, a cytochrome of interest because
it is more highly expressed during growth on Fe(III), inhibited Fe(III)
reduction (Butler et al., 2004), but deletion of macA was also associated
with a lack of OmcB (Kim and Lovley, 2008).
Another cytochrome of interest is PgcA, which is upregulated during
growth on Fe(III) oxide (Ding et al., 2008; Holmes et al., 2011b). Selection
for enhanced growth of G. sulfurreducens on Fe(III) oxide selected for
mutations that increased expression of PgcA (Tremblay et al., 2011b).
Further, PgcA is a member of one of the cytochrome families conserved
across several Geobacter species (Butler et al., 2010b). Further study of
this cytochrome is underway.
The putative multicopper protein, OmpB, which is localized to the outer
surface of G. sulfurreducens (Qian et al., 2007), also appears to be involved
in the reduction of Fe(III) oxide but is not required for the reduction of
soluble Fe(III) (Mehta et al., 2006). The pseudo-pilin OxpG is required
for OmpB export. The OmpB homolog, OmpC, is also important for opti-
mal Fe(III) oxide reduction but has not been experimentally localized and
has different expression patterns than OmpB (Holmes et al., 2008).

Homologs with four copper-binding sites, two at the N-terminus and two
at the C-terminus, are found in all of the Geobacter genomes, though the
protein size ranges from ca. 800 to 1700 aa (Butler et al., 2010b). Phyloge-
netically, the omp genes form two distinct clades, the B-type and the C-
type, and not all genomes contain both types (Holmes et al., 2008). No
homologs were found in the two Pelobacter genomes. Various potential
roles for OmpB and OmpC have been suggested (Holmes et al., 2008;
Mehta et al., 2006), but purification and characterization of the proteins
are required to better evaluate these possibilities.
The many other underexplored cytochromes and other putative redox-
active proteins in G. sulfurreducens warrant further study, as do proteins likely
to be involved in cytochrome export (Afkar et al., 2005), and the cytochromes
in other Geobacter species. For example, G. uraniireducens increases the tran-
scriptional expression of several cytochromes when cultured in anoxic
sediments versus growth on soluble electron acceptors (Holmes et al., 2009).
Additional structural studies will provide important insights into their function
(Londer et al., 2002, 2006a,b; Morgado et al., 2007, 2009; Pessanha et al., 2004,
2006; Pokkuluri et al., 2004, 2008, 2009, 2011).
Development of genetic systems for Geobacter species other than
G. sulfurreducens will also aid in functional analysis, as will the approach
of determining which cytochrome functions can be completed in mutants
of G. sulfurreducens with cytochrome gene sequences from other
Geobacter species (Yun et al., 2011a).
7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide
Several models have been advanced for how Geobacter species transfer
electrons to insoluble Fe(III) oxides. A miscalibrated spectrophotometer
in initial studies with G. metallireducens (Gorby and Lovley, 1991) resulted
in the mistaken suggestion that b-type cytochrome(s) were important in
extracellular electron transfer, but subsequent studies demonstrated a role
for c-type cytochromes in the reduction of Fe(III) and other metals
(Lovley et al., 1993a). An early model for Fe(III) oxide reduction by
Geobacter sulfurreducens suggested that it released a low-molecular-weight
c-type cytochrome, which acted as an electron shuttle between cells and
Fe(III) oxide (Seeliger et al., 1998). However, this concept was refuted in
a number of studies, including studies in the laboratory, which initially
developed the electron shuttling concept (Lloyd et al., 1999; Nevin and
Lovley, 2000; Straub and Schink, 2003).
Evidence consistent with the need for direct contact is the lack of Fe(III)
reduction when cells are separated from Fe(III) oxide contained within
microporous alginate beads (Nevin and Lovley, 2000) or agar (Straub
and Schink, 2003). This was observed with G. metallireducens (Nevin
and Lovley, 2000) as well as G. sulfurreducens, G. bremensis, and
G. pelophilus (Straub and Schink, 2003). In contrast, Shewanella (Nevin
and Lovley, 2002b) and Geothrix (Nevin and Lovley, 2002a) species, and
Fe(III)-reducing enrichment cultures (Straub and Schink, 2003), produced
shuttles that permitted reduction of Fe(III) oxide at a distance. Further, G.
metallireducens also did not appear to produce chelators that could solubi-
lize Fe(III), whereas Shewanella (Nevin and Lovley, 2002b) and Geothrix
(Nevin and Lovley, 2002a) species did solubilize Fe(III) under similar
conditions.
Although some of the components that appear to be involved in elec-
tron transfer to Fe(III) oxides have been identified, the understanding of
how these, and potentially other components, fit together is far from com-
plete. As noted above, OmcS is likely to have an important role in Fe(III)
oxide reduction because (1) OmcS expression is highly upregulated during
growth on Fe(III) oxide (Ding et al., 2008; Holmes et al., 2011b; Mehta
et al., 2005); (2) gene deletion studies indicate that the OmcS is required
for Fe(III) oxide reduction (Mehta et al., 2005); (3) OmcS is specially
associated with pili (Leang et al., 2010), which, as described above, are
electrically conductive and are required for Fe(III) oxide reduction; and
(4) purified OmcS can transfer electrons to Fe(III) oxide and may bind
Fe(III) (Qian et al., 2011). The simplest explanation for these observations
is that electrons that are transported along the pili are transferred to Fe
(III) oxide via OmcS. There is no obvious route for electrons to get to
OmcS other than the pili and the lack of Fe(III) reduction in the absence
of OmcS suggests that electrons cannot be directly transferred from the pili
to Fe(III) oxide.
There is little information on how electrons are transferred to the pili.
This could conceivably take place in the periplasm, or even the inner mem-
brane, but the requirement for OmcB, which is located in the outer mem-
brane, suggests that electron transfer near the outer surface of the cell is
more likely. The fact that OmcB is embedded in the outer membrane
suggests that it might be difficult for OmcB and pili to associate closely
enough for electron transfer between the two. The need to mediate elec-
tron transfer from OmcB to the pili at the outer cell surface may explain
why other potentially redox-active outer-surface components, such as
other c-type cytochromes and the putative multicopper proteins OmpB
and OmpC, are important in Fe(III) oxide reduction.
The role of other outer-surface cytochromes in Fe(III) oxide reduction is

also not completely understood. OmcE can be an abundant c-type cyto-
chrome under some growth conditions, but cells can eventually overcome
deletion of omcE and reduce Fe(III) oxide (Mehta et al., 2005). It has been
proposed that OmcZ localized in an extracellular matrix could be important
in Fe(III) oxide reduction (Rollefson et al., 2011), but this is not consistent
with several observations including (1) OmcZ is not required for Fe(III)
oxide reduction (Nevin et al., 2009), (2) low expression of omcZ in cells
growing on Fe(III) oxide (Holmes et al., 2011b), and (3) purified OmcZ only
poorly reduces Fe(III) oxide (Inoue et al., 2010).
If OmcB is the conduit for electrons out of the cell and toward pili, then
the next question is what is the electron donor for OmcB? Periplasmic
cytochromes are potential sources, ferrying electrons from the inner
membrane to the outer membrane. As noted above, a number of periplasmic
c-type cytochromes have been identified in G. sulfurreducens, but no electron
transfer link between these cytochromes and OmcB, or any other electron
acceptor, has been documented.
Diagrams for how the electrons may flow to Fe(III) oxide from
G. sulfurreducens are available (Lovley, 2011c), but clearly we are still at
the hypothesis stage and more research on electron transfer out of the cell
is warranted. Novel strategies for elucidating important components are
likely to be helpful. For example, adaptive evolution for improved Fe(III)
oxide reduction in G. sulfurreducens provided further evidence for the
importance of pili in Fe(III) oxide reduction as well as identifying an addi-
tional c-type cytochrome that may be involved (Tremblay et al., 2011b).
Studies on species other than G. sulfurreducens are also warranted to
look for commonalities that are general features of electron transfer to
Fe(III) oxides in all Geobacter species. For example, unique PilA
sequences are conserved in Geobacter species (Reguera et al., 2005) and
recent gene deletion studies have demonstrated that PilA is required for
Fe(III) oxide reduction in G. metallireducens (Tremblay et al., 2011a).
In contrast, outer-surface cytochromes sequences are poorly conserved in
Geobacter species (Butler et al., 2010b), suggesting that there is less specific-
ity in cytochrome requirements. However, there is still an opportunity to
look for commonality in mechanisms. For example, if electrons cannot be
directly transferred from pili to Fe(III) oxides, then it would be expected
that G. metallireducens, which does not have an OmcS homolog (Butler
et al., 2010b), would possess another cytochrome, which like OmcS, is
associated with pili and necessary for Fe(III) oxide reduction.
Additional research is also required on the early steps of electron trans-
fer across the inner membrane and to the electron carriers responsible for
the terminal steps in electron transfer to Fe(III) and other extracellular

electron acceptors. Although possible electron carriers can be identified
from genome sequences, experimental studies are required before defini-
tive models can be developed. One of the key features of extracellular
electron transfer in Geobacter species is the poor energy yields available
from this mode of respiration in comparison with the reduction of soluble
electron acceptors within the cell (Esteve-Nunez et al., 2004, 2005;
Mahadevan et al., 2006). This can be attributed, at least in part, to the fact
that intracellular reduction of electron acceptors consumes protons along
with electrons, but when electrons are transferred out of the cell, this pro-
ton sink is lost, requiring export of protons that does not contribute to the
development of a proton-motive force across the inner membrane
(Mahadevan et al., 2006, 2011).
7.4. Model for Extracellular Electron Transfer to Electrodes
Like Fe(III) oxide, electrodes represent an insoluble, extracellular electron

acceptor. Initial studies with G. sulfurreducens suggested that it did not
produce electron shuttles in order to promote electron transfer to
electrodes (Bond and Lovley, 2003) and electrochemical studies supported
this conclusion (Busalmen et al., 2008, 2010; Marsili et al., 2008, 2010;
Marsili et al.; Richter et al., 2009). This is consistent with the similar con-
cept of direct electron transfer to Fe(III) oxide.
However, there are major differences between the electrodes and Fe
(III) oxide because electrodes function as stable long-term electron
acceptors, whereas once Fe(III) is reduced in one location cells need to
find additional sources of Fe(III). The stability of the electrode as an elec-
tron acceptor makes it possible for Geobacter to produce thick (> 50 mm)
biofilms on electrodes (Franks, 2010; Franks et al., 2009; Nevin et al.,
2009; Reguera et al., 2006), which are not formed during growth on Fe(III)
oxide. Thus, the necessity to transfer electrons through a biofilm may
require different electron transport strategies and may place different
selective pressures on cells.
Fashioning one coherent model for electron transfer from
G. sulfurreducens to electrodes that can accommodate all the data avail-
able in the literature is difficult. There is substantial confusion in the liter-
ature because models generated from preliminary data are often ruled out
as more data becomes available. For example, early studies in our labora-
tory investigated electron transfer in systems producing relatively low
amounts of current in which most of the cells were closely associated with
the anode surface. Under those conditions, OmcS was highly expressed
and was essential for current production (Holmes et al., 2006). In contrast,
in subsequent studies with systems producing much more current, OmcS
was not highly expressed and cells adapted to produce current comparable
to that of wild type when OmcS was deleted (Nevin et al., 2009). Rather,
OmcZ was highly expressed in the high-current density biofilms. OmcZ
and OmcS do not appear to have equivalent functions, based on their dif-
ferent localization and other factors, and it is generally the case that when
OmcS is highly expressed OmcZ expression is low and vice versa. The
geometry of the electrode material may also influence gene expression
patterns, and presumably electron transfer pathways, with expression
patterns on graphite fiber electrodes resembling more closely the expression
in the early low-current density biofilms on planar graphite surfaces (K.P.
Nevin et al., unpublished data). Therefore, instead of attempting to develop
one universal model for electron transfer to electrodes, we have focused on
electron transfer in thick (> 50 mm) electrode biofilms, which produce
high-current densities, because a major goal is to understand the production
of high-current densities in order to further optimize current output.
An initial observation in the development of higher current densities was
that the increase in current was proportional to the increase in biomass on
the anode, suggesting that cells at great distance from the anode were con-
tributing to current production (Reguera et al., 2006). Subsequent studies
have confirmed the high metabolic activity of such cells (Franks et al.,
2010b). The finding that deleting pilA prevented high-current densities led
to the hypothesis that networks of pili in the G. sulfurreducens biofilms con-
ferred conductivity on the biofilm and a route for electrons released from
cells at distance to be transported to the electrode (Reguera et al., 2006).
Consistent with this concept, modeling studies indicated that the high-
current density in microbial fuel cells would be feasible only if Geobacter
biofilms were assumed to be electrically conductive (Marcus et al., 2007;
Torres et al., 2008, 2009b). However, the suggestion that biofilms of Geo-
bacter species could be conductive contrasted with previous studies, which
had demonstrated that the biofilms of bacteria act as insulators (Dheilly
et al., 2008; Herbert-Guillou et al., 1999; Muoz-Berbel et al., 2006).
Measurement of the conductance of viable G. sulfurreducens biofilms with a
novel two-electrode system revealed that the biofilms that had been grown
with an electrode as the electron acceptor had remarkable conductivity,
comparable to that of synthetic organic conducting polymers, such as poly-
aniline and polyacetylene (Malvankar et al., 2011b). In contrast, biofilms
grown in the same system, but with fumarate as the electron acceptor, had
low conductivity. The biofilms of Escherichia coli and Pseudomonas
aeruginosa were not conductive. Evaluation of strains of G. sulfurreducens

with different biofilm conductivities demonstrated a strong correlation
between the abundance of PilA in the biofilm and conductivity, suggesting that
the conductivity was related to the extent of pilin production.
The temperature dependence of biofilm conductivity was similar to that
of pilin preparations, demonstrating a metallic-like conduction mechanism,
which was further confirmed with electrochemical gating studies
(Malvankar et al., 2011b). These results suggested that the biofilm conduc-
tivity was related to the metallic-like conductivity of the pilin network. None
of these results support the concept of electron hopping through biofilms via
c-type cytochromes. Further, denaturing the c-type cytochromes in the bio-
films had no impact on conductance and there was no correlation between
conductance and cytochrome content of the biofilms. These results suggest
that the novel metallic-like conductivity in G. sulfurreducens can be
attributed to the surprising metallic-like conductivity of its pilin networks.
Consistent with the apparent importance of pili in conduction of
electrons through G. sulfurreducens biofilms, the gene for PilA is among
the most highly upregulated genes in current-producing biofilms (Nevin
et al., 2009). Selective pressure for enhanced current production yielded a
strain of G. sulfurreducens that produced more pili (Yi et al., 2009).
Deletion of pilA significantly inhibited current production, with only cells
near the electrode surface remaining metabolically active (Reguera et al.,
2006). Although the pilin constructed of PilA may have a structural role
in biofilm formation under some conditions (Reguera et al., 2007), the pilA
deletion mutant readily formed thick biofilms on the graphite electrode
material if fumarate was provided as an alternative electron acceptor
(Nevin et al., 2009).
The concept of electron transport through G. sulfurreducens biofilms via
conductive pilin networks contrasts with many studies that have suggested
that more traditional electron transfer via cytochromes moves electrons
through the biofilms. Biofilms of wild-type G. sulfurreducens growing on
electrodes are visibly red, due to the cytochrome abundance. Many studies
have provided evidence that cytochromes are oxidized and reduced in G.
sulfurreducens biofilms in electrical contact with electrodes (Esteve-Nez
et al., 2011; Fricke et al., 2008; Jain et al., 2011; Liu et al., 2010b, 2011;
Marsili et al., 2008, 2010; Millo et al., 2011; Richter et al., 2009; Srikanth
et al., 2008; Strycharz et al., 2011b), but the interpretation that this
represents electron transfer through the biofilm by electron hopping via
c-type cytochromes in analogy with redox hydrogels (Heller, 2006; Richter
et al., 2009) is not consistent with the studies (Malvankar et al., 2011b) on
biofilm conductance.
The likely explanation for this apparent discrepancy is that the electro-
chemical analyses only probed the biofilm-electrode interface and not the
entire biofilm (Dumas et al., 2008a; Franks et al., 2010a). The cytochromes
at the interface may function as an electrochemical gate, promoting
electron transfer to the electrode surface (Dumas et al., 2008a).
A likely candidate for a cytochrome functioning as an electrochemical
gate is the outer-surface c-type cytochrome OmcZ. The OmcZ gene is one
of the most highly upregulated genes in current-producing cells, and if omcZ
is deleted, the cells produce low levels of current (Nevin et al., 2009). There is
much higher resistance for electron transfer to electrodes in cells lacking
OmcZ, which was originally interpreted as OmcZ conferring conductivity
throughout the biofilm (Richter et al., 2009). However, this cannot be correct
as the conductance of biofilms of a strain with lower abundance of OmcZ was
higher than those of wild type (Malvankar et al., 2011b). Further, cells
throughout the biofilm express omcZ (Franks et al., 2011). OmcZ
accumulates at the biofilm-electrode interface, consistent with the electro-
chemical gate hypothesis (Inoue et al., 2011).
The reason that OmcZ or other cytochromes might be required to facili-
tate current production is that a significant energy barrier might exist across
the biofilm-electrode interface similar to a semiconductormetal interface
(Lange and Mirsky, 2008). The wide range of reduction potentials ( 420
to 60 mV) of the multiple hemes in OmcZ (Inoue et al., 2010) might help
overcome this energy barrier in a manner similar to electrochemical gating
in molecular electronics (Vanmaekelbergh et al., 2007).
7.5. Extracellular Electron Transfer in Syntrophy
The finding that key elements of extracellular electron exchange in

G. sulfurreducens were required for effective electron exchange between
G. metallireducens and G. sulfurreducens and the finding that the
aggregates were electrically conductive (Summers et al., 2010) suggest that
components of these cells can form an electrically conductive matrix which
permits direct electron exchange between the partners (Lovley, 2011a,c).
OmcS was very abundant in the aggregates, which was attributed to a
mutation in a regulatory gene of G. sulfurreducens that was selected
for during adaption for ethanol metabolism. Introducing a strain of
G. sulfurreducens with the regulator inactivated hastened adaption for eth-
anol metabolism, whereas deleting the gene for OmcS or PilA prevented
aggregate formation and ethanol metabolism. These results suggest that
electron transfer through OmcS and the pili of G. sulfurreducens is an

important part of the interspecies electron exchange.
Which components of G. metallireducens are important for the electron
exchange are not known, but this question should now be addressable
because of the recent development of methods for genetically man-
ipulating G. metallireducens (Tremblay et al., 2011b). The study of direct
electron transfer from electrodes to cells (Strycharz et al., 2011a) may help
elucidate the electron-receiving component of the model.
It is important to recognize that aggregation of the two syntrophic
partners may not be a prerequisite for direct interspecies electron transfer.
In a manner similar to the mechanisms proposed for Fe(III) oxide reduc-
tion, Geobacter species could establish temporary contact with a recipient
cell, offload electrons, and then move on.
7.6. Model for Extracellular Electron Transfer to Other

Extracellular Electron Acceptors
The display of multiple low-potential c-type cytochromes on the outer sur-

face of Geobacter species confers the capacity to reduce a wide diversity of
soluble electron acceptors at the outer cell surface. Reduction of these
electron acceptors may be rather nonspecific. For example, deleting the
genes for individual outer-surface cytochromes only partially inhibited
the ability of G. sulfurreducens to reduce humic substances and AQDS.
Only when the genes for OmcB, OmcE, OmcS, OmcT, and OmcZ were
deleted in the same strain was humic substance and AQDS reduction
eliminated (Voordeckers et al., 2010).
Although the final product of U(VI) reduction is U(IV), the initial
reduction of U(VI) may be a one electron transfer followed by dispropor-
tionation of U(V) to U(VI) and U(IV) (Renshaw et al., 2005). Initially it
was considered that U(VI) might be reduced in the periplasm (Lloyd
et al., 2002), but the accumulation of uranium in the periplasm that was a
main line of evidence for periplasmic reduction was later found to be an
artifact (Shelobolina et al., 2007a). Systematic deletion of the genes for
the most abundant outer-surface c-type cytochromes in a study comparable
to one on reduction of humic substances has indicated that the site of
reduction is the outer surface of the cell (R. Orellana, unpublished data).
Purified OmcZ (Inoue et al., 2010) and OmcS (Qian et al., 2011) reduce
U(VI), and it is likely that many low-potential c-type cytochromes will
be capable of U(VI) reduction (Lovley et al., 1993b). It seems likely that
the other metallic ions that Geobacter species can reduce may also be
reduced in a similar nonspecific manner.
In vitro studies with the abundant periplasmic c-type cytochrome of the
closely related Desulfuromonas acetoxidans demonstrated that this cyto-
chrome could reduce elemental sulfur in vitro (Pereira et al., 1997) and
periplasmic reduction of sulfur has been a model. However, systematic
reduction of the outer-surface c-type cytochromes of G. sulfurreducens
has suggested that elemental sulfur is also reduced at the outer cell surface
(S. Dar, unpublished results).
7.7. Capacitor Role of Cytochromes
In addition to their role in extracellular electron transfer discussed above,

and possible environmental sensing discussed later, the abundant c-type
cytochromes of Geobacter species may have the additional role of function-
ing as capacitors for electron storage under some environmental conditions
(Esteve-Nunez et al., 2008; Lovley, 2008a). The sheer abundance of c-type
cytochromes in cells growing under electron-acceptor-limiting conditions
or in biofilms producing electrical current makes the cultures/biofilms visi-
bly red. The capacity to store electrons in periplasmic and outer-surface
cytochromes may be beneficial for Geobacter species because Fe(III)
sources are heterogeneously dispersed and there are likely to be periods
when Geobacter species are not in direct contact with Fe(III) oxides. In
fact, when Geobacter species are most actively growing during in situ ura-
nium bioremediation they are highly planktonic and thus periodically out
of contact with Fe(III) (Anderson et al., 2003; Dar et al., 2011; Kerkhof
et al., 2011). Cytochromes positioned beyond the inner membrane, in the
oxidized state, can accept electrons from inner membrane electron carriers,
permitting continued respiration even when Fe(III) is not available.
Energy conservation under such conditions is expected to be similar to
when Fe(III) is available because conservation of energy results from elec-
tron transfer components in the inner membrane. Subsequent steps in elec-
tron transport to extracellular electron acceptors do not conserve
additional energy for the microorganism; they are just necessary in order
to provide electron acceptors for electron transfer across the inner mem-
brane. Once cells contact Fe(III), the electrons stored in the cytochromes
can be discharged.
Another observation consistent with this concept is that G. meta-
llireducens specifically expresses flagella when grown on insoluble Fe(III)
or Mn(IV) oxides, but not when grown on soluble Fe(III) citrate (Childers
et al., 2002). Deleting a gene for flagella production inhibited Fe(III) oxide
reduction in G. metallireducens, but not the reduction of Fe(III) citrate
(Tremblay et al., 2011a). Changing the sequence of a gene for a master
regulator of flagella to confer motility in the otherwise nonmotile
G. sulfurreducens enhanced Fe(III) oxide reduction (Ueki et al., 2011).
The negative impact on Fe(III) reduction of making G. metallireducens
nonmotile was stronger when the cells were grown with sediment Fe(III)
oxides as the electron acceptor than in cultures with synthetic Fe(III)
oxides, consistent with the greater dispersal of Fe(III) oxides in sediments
(Tremblay et al., 2011a). Chemotaxis might guide Geobacter species to
Fe(III) and Mn(IV) oxide sources (Childers et al., 2002).
Studies with current-producing biofilms of G. sulfurreducens have con-
firmed this inference of c-type cytochromes conferring capacitance
(Malvankar et al., 2011a). The biofilms had capacitance comparable to that
of synthetic supercapacitors. Multiple lines of evidence demonstrated that
the biofilm capacitance could be attributed to the c-type cytochromes. As
discussed below, this novel form of capacitance may be a useful contribu-
tion to the field of bioelectronics.
8. REGULATION OF METABOLISM
In order to understand how Geobacter species function in diverse

environments, and how they are likely to change their metabolism in response
to changes in environmental conditions, it is important to understand
how gene expression is regulated. The elucidation of regulatory networks in
Geobacter species is in its infancy, but some progress has been made.
8.1. Sigma Factors
Sigma factors play a key role in the regulation of gene expression in

response to changing environments, and bacteria typically employ multiple
sigma factors to optimize their responses. Each species of sigma factors
recognizes specific promoter elements of a certain set of genes and initiates
their transcription. The genome of G. sulfurreducens encodes homologs of
RpoD (s70), RpoS (sS, s38), RpoH (sH, s32), RpoN (sN, s54), RpoE
(sE, s24), and FliA (RpoF, sF, s28) found in E. coli and many other bacteria
(Mth et al., 2003). In other bacteria, RpoD is generally the major sigma
factor for most housekeeping genes (Ishihama, 2000). Alternative sigma
factors are required for stress response genes. For example, RpoS is
required for general stress response genes including stationary-phase genes.
RpoH is necessary for heat-shock genes. RpoN is involved in transcription
of genes for nitrogen deficiency and some other stresses. FliA participates in
regulation of flagella and chemotaxis genes. RpoE represents
extracytoplasmic function (ECF) sigma factors, which are involved in regula-
tion of genes for outer membrane or periplasmic proteins.
The RpoD homolog appears to be the major sigma factor in G.
sulfurreducens as transcriptomic analysis has demonstrated that a large
number of genes were transcribed by RNA polymerase containing RpoD
(Qiu et al., 2010). It is likely that G. sulfurreducens RpoD recognizes pro-
moter elements similar to those of other bacterial RpoD homologs (Qiu
et al., 2010; Yan et al., 2006). However, genetic and biochemical studies
for RpoD have not been conducted.
The RpoS homolog is the stationary-phase sigma factor in G.
sulfurreducens (Nunez et al., 2004). It is also involved in response to oxygen
exposure and in growth with oxygen as the electron acceptor. An rpoS-dele-
tion mutant exhibited less viability at the stationary phase than the wild-type
strain and slower recovery after exposure to oxygen. The mutant was also
defective in reduction of insoluble Fe(III) oxide but not of soluble Fe(III)
citrate. Transcriptome and proteome analyses revealed genes in a variety
of cellular functions under the control of G. sulfurreducens RpoS, which
include oxidative stress and nutrient limitation response genes and genes
for c-type cytochromes (Nunez et al., 2006). Among the c-type cytochromes
is MacA, which is known to be critical for Fe(III) reduction (Butler et al.,
2004). It appears that promoters recognized by RpoS in G. sulfurreducens
are similar to those recognized by RpoD, as found in other bacteria (Yan
et al., 2006). However, genes regulated by RpoS in G. sulfurreducens are
diversified from those in other bacteria (Santos-Zavaleta et al., 2011). These
studies suggest that the RpoS homolog plays important roles in
G. sulfurreducens under conditions that Geobacter species typically encoun-
ter in subsurface environments.
RpoH is the heat-shock sigma factor in G. sulfurreducens (Ueki and Lovley,
2007). Expression of the rpoH gene was induced by heat shock from 30 to
42 C and appears to be controlled by RpoH itself as well as the HrcA/CIRCE
system. HrcA is known to be a repressor for heat-shock genes by binding the
CIRCE (Controlling Inverted Repeat of Chaperon Expression) element in
other bacteria (Schulz and Schumann, 1996; Zuber and Schumann, 1994).
An rpoH-deletion mutant of G. sulfurreducens was unable to grow at 42 C,
whereas the wild-type strain could. The expression of heat-shock response
genes decreased dramatically in the rpoH-deletion mutant.
In contrast to most other bacteria for which RpoN is dispensable under

some conditions, the RpoN homolog appears to be a vital sigma factor in
G. sulfurreducens (Leang et al., 2009). Transcriptome analysis demonstrated
that RpoN regulates a large number of genes involved in a wide range of cel-
lular functions including those encoding enzymes for ammonia assimilation,
which are predicted to be essential under all growth conditions in
G. sulfurreducens. RpoN also regulates genes that were shown to be important
for growth in subsurface environments or electricity production in microbial
fuel cells, such as flagella biosynthesis, pili biosynthesis, and c-type
cytochromes. Promoter elements recognized by the G. sulfurreducens RpoN
are highly similar to those recognized by other bacterial RpoN homologs.
Transcription initiation by RNA polymerase containing RpoN requires an
enhancer-binding protein, and the G. sulfurreducens genome encodes more
putative transcription factors in the enhancer-binding protein family than most
bacteria (Karlin et al., 2006; Mth et al., 2003). Thus, the RpoN homolog is a
global regulator controlling a complex transcriptional network modulating
physiological responses in G. sulfurreducens.
The FliA homolog appears to regulate flagella and chemotaxis genes in
G. sulfurreducens as analysis of the G. sulfurreducens genome identified
sequences similar to those recognized by other bacterial FliA homologs
(Leang et al., 2009; Tran et al., 2008). As noted elsewhere, flagellar motility
and chemotaxis are likely to be important for growth in subsurface
environments. However, the function of the FliA homolog has not been
experimentally studied in Geobacter species.
The RpoE homolog has not been characterized in Geobacter species.
RpoE homologs or ECF sigma factors have been shown to control a vari-
ety of cellular functions in other bacteria. Amino acid sequences of ECF
sigma factors appear to be more diverse than those of other families of
sigma factors (e.g., RpoD, RpoS, RpoH, RpoN, and FliA). Therefore, it
is difficult to predict the function of the RpoE homologs in Geobacter spe-
cies solely by their sequences. Whereas homologs of RpoD, RpoS, RpoH,
RpoN, and FliA are highly conserved among Geobacter species whose
genome sequences are available, RpoE homologs are not, suggesting that
RpoE might be involved in regulation of species-specific features.
8.2. Transcription Factors
Transcription factors generally regulate genes for more specific cellular

functions than sigma factors and further fine-tune gene regulation in
response to environmental and physiological changes. Transcription
factors include an activator and a repressor, which promote or inhibit tran-

scription by RNA polymerase, respectively. Some transcription factors can
function as both an activator and a repressor. The G. sulfurreducens genome
encodes 151 putative transcription factors (Mth et al., 2003). Transcription
factors classified as a response regulator on the basis of sequence similarity
are described in the section on two-component systems below.
The novel transcriptional repressor, HgtR, is induced in G.
sulfurreducens when hydrogen is available as an electron donor and
represses expression of citrate synthase and other genes encoding enzymes
involved in central metabolism (Ueki and Lovley, 2010a). HgtR also
regulates a gene encoding a putative transcription factor in the GntR fam-
ily. Target genes of this GntR homolog in Geobacter species have not been
identified. A transcription factor in the RpoN-dependent enhancer-binding
protein family appears to regulate expression of hgtR, which has sequences
highly similar to the RpoN recognition consensus sequences in its pro-
moter region. Further, a gene encoding an enhancer-binding protein is
located upstream of the hgtR homologs in Geobacter species. These
enhancer-binding protein homologs contain a domain similar to the C-ter-
minal domain of the iron-only hydrogenase large subunit at the N-termi-
nus. It is possible that these enhancer-binding protein homologs sense
environmental and/or intracellular hydrogen and activate the hgtR
homologs. Therefore, it is likely that a novel regulatory cascade mediated
by multiple transcription factors genes controls expression of central
metabolism genes in Geobacter species.
Studies on the adaption of G. sulfurreducens to grow on lactate revealed
a transcriptional regulator that regulates expression of the genes for succi-
nyl-CoA synthetase (Summers et al., 2011), a TCA cycle enzyme that is
required for growth on lactate, but not acetate (Galushko and Schink,
2000; Segura et al., 2008). The G. sulfurreducens gene GSU0514, which is
a homolog of the IclR transcription factor, encodes a transcriptional
repressor for the succinyl-CoA synthetase subunit genes sucC and sucD
(Summers et al., 2011). Mutations in GSU0514 were selected for during
adaption for enhanced growth on lactate, which enhanced expression of
sucC and sucD and promoted lactate metabolism (Summers et al., 2011).
Another adaptive evolution study identified the transcription factor,
GSU1771, which controls the expression of genes that are important for
Fe(III) oxide reduction (Tremblay et al., 2011b). GSU1771 is a homolog
of Streptomyces antibiotic regulatory protein (SARP) (Wietzorrek and
Bibb, 1997). Adaption of G. sulfurreducens for more rapid growth on Fe
(III) oxide yielded strains that accumulated a mutation that interrupted
the GSU1771 gene. Inactivation of GSU1771 in the wild-type strain
enhanced the ability to reduce Fe(III) oxide and increased the expression of
the gene for PilA, the structural protein for the electrically conductive pili.
Although dissimilatory metal reduction by Geobacter species has been
extensively studied, effects of the availability of metals for assimilatory
purposes on growth and activity of Geobacter species have gained less
attention. For instance, Fe(II) appears to play a critical physiological role
in Geobacter species as they contain an unusually large number of iron-sul-
fur proteins such as c-type cytochromes, which have been shown to be
essential for dissimilatory metal reduction. The Fe(II)-dependent tran-
scription factor, Fur, is an important regulator for Fe(II) influx in other
bacteria (Escolar et al., 1999) and all available Geobacter genomes contain
a cluster consisting of homologs of fur, as well as feoB, which encodes an
iron uptake protein and ideR, another Fe(II)-dependent transcription fac-
tor (O'Neil et al., 2008). In chemostat cultures, the expression of the fur-
feoB-ideR cluster decreased as Fe(II) concentrations increased, suggesting
that transcript abundance could serve as an indication of limitation of iron
for assimilation. Monitoring transcript abundance of the Geobacter species
in groundwater surprisingly revealed that iron availability might be limiting
under some bioremediation conditions (O'Neil et al., 2008). Analyses of
Geobacter genomes identified sequences in feoB and other genes that are
similar to other bacterial Fur-binding sites, suggesting that Fur controls
feoB in Geobacter species.
A number of biological processes in energy generation, nitrogen assimi-
lation, and detoxification require nickel-dependent enzymes such as
hydrogenase, carbon monoxide dehydrogenase, and urease (Mulrooney
and Hausinger, 2003; Zhang et al., 2009). The Ni(II)-dependent transcrip-
tion factor NikR is known to regulate nickel transporters in other bacteria
(Chivers and Sauer, 1999; Wang et al., 2009). The G. uraniireducens NikR
homolog was shown to bind the promoter regions of two different genes,
nik(MN)1 and nik(MN)2, which encode ABC-type transporters (Benanti
and Chivers, 2010). The DNA-binding mode of the G. uraniireducens
NikR homolog was distinct from other members of the NikR family.
Geobacter species are likely to encounter oxygen intrusions in subsur-
face environments, particularly at the oxic/anoxic interface where Fe(III)
sources are abundant. Thus, the ability of Geobacter species to tolerate
exposure to low concentrations of oxygen and even grow with oxygen as
the terminal electron acceptor may be a critical factor to survival in subsur-
face environments (Lin et al., 2004; Mouser et al., 2009a). Many bacterial
cells are equipped with oxidative responsive systems, which are regulated
by RecA and LexA (Butala et al., 2009; Cox, 2007). LexA is a transcription
factor controlling genes in the SOS system. The G. sulfurreducens genome
encodes two independent LexA homologs, which appear to be

autoregulated (Jara et al., 2003). Unlike other bacterial LexA, G.
sulfurreducens LexA homologs may not control recA and other genes
known to be involved in the SOS system because sequences similar to G.
sulfurreducens LexA-binding sites located in the G. sulfurreducens lexA
genes are absent from these SOS system genes in G. sulfurreducens. Thus,
G. sulfurreducens may employ unique regulatory mechanisms in oxidative
responsive systems.
Transcriptional regulators control the expression of genes involved in
the degradation of aromatic compounds. BgeR is a transcriptional repres-
sor that regulates genes for the metabolism of aromatic compounds as well
as another transcription factor involved in aromatic metabolism in
G. bemidjiensis (Ueki, 2011). BgeR belongs to the Rrf2 family, but the sim-
ilarity is limited to the N-terminal region, which is likely a DNA-binding
domain. Its C-terminal region does not show similarity to known proteins
except for BgeR homologs in other Geobacter species. It is likely that
genes for aromatic compound degradation are controlled by regulatory
cascades consisting of multiple transcription factors in Geobacter species.
This is a topic that warrants further study because of its potentially
important role in bioremediation.
8.3. Two-Component Systems
Geobacter species have one of the highest IQs of bacteria, which is a mea-
sure of the adaptive potential of an organism on the basis of the total num-
ber of signaling proteins including the two-component system encoded in a
given genome (Galperin, 2005). The genomes of Geobacter species encode
an unusually large number of genes for the two-component signaling
proteins (Aklujkar et al., 2009, 2010; Mth et al., 2003). The two-component
system typically consists of a sensor histidine kinase, which senses environ-
mental signals, and a response regulator, which generally influences the gene
expression necessary for the adaptation (Egger et al., 1997). The two compo-
nents are often colocalized in the same operon in other bacteria (Mizuno,
1997), but this is often not the case in Geobacter species. Most of the puta-
tive sensor domains of the two-component systems in Geobacter species
are unique or show similarity to uncharacterized systems in other bacteria.
Some of the sensor domains have a c-type heme-binding motif (Londer
et al., 2006b; Pokkuluri et al., 2008) and may participate in redox control
of complex biological processes in Geobacter species.
An important response regulator is PilR, which is an RpoN-dependent

enhancer-binding protein that regulates expression of pilA, the gene for
the structural pilin protein (Juarez et al., 2009). The histidine kinase PilS
appears to regulate PilR because pilS is immediately upstream of pilR.
Transcriptomic and bioinformatic approaches identified a number of genes
including those for c-type cytochromes, such as OmcB and OmcS, under
the control of PilR (Juarez et al., 2009; Krushkal et al., 2010). Adaptive evo-
lution for syntrophic growth of G. metallireducens and G. sulfurreducens
selected for a strain of G. sulfurreducens with a mutation in pilR, which
enhanced production of the c-type cytochrome OmcS (Summers et al., 2010).
Novel regulatory cascades consisting of two two-component systems reg-
ulate nitrogen-fixation gene expression in G. sulfurreducens (Ueki and
Lovley, 2010b). GnfM, a member of the enhancer-binding protein family,
contains a receiver domain at the N-terminus and thus is also a response
regulator of a two-component system in which the activity of GnfM
appears to be regulated by the histidine kinase GnfL. The GnfM gene
seems to be essential for growth, probably because it regulates the expres-
sion of genes involved in nitrogen metabolism that are important even
when ammonium is present (Leang et al., 2009). In addition to the nitrogen
metabolism genes, the GnfL/GnfM system activates genes encoding regu-
latory proteins of a two-component system during nitrogen fixation that
comprises the histidine kinase GnfK and the response regulator GnfR,
which has an RNA-binding domain. GnfK modulates the activity of GnfR
by phosphorylation. Phosphorylated GnfR exhibits RNA-binding activity.
The GnfK/GnfR system regulates by transcription antitermination the
expression of a subset of the nitrogen-fixation genes, such as nifH, nifEN,
nifX, glnK, and amtB, whose transcription is activated by the GnfL/GnfM
system and whose promoter region contains transcription termination
signals. The GnfK/GnfR system plays a critical role in the nitrogen-fixation
gene regulation as deletion mutants of gnfK or gnfR are defective in
growth dependent on nitrogen fixation.
Expression of the gene bamY, which encodes benzoate-CoA ligase, is
induced during growth of G. metallireducens on benzoate (Butler et al.,
2007; Wischgoll et al., 2005). The bamV and bamW genes encoding a puta-
tive histidine kinase and a putative response regulator, respectively, are
located in the vicinity of the bamY gene (Wischgoll et al., 2005). Aromatic
compounds induced the expression of the bamV gene (Butler et al., 2007;
Wischgoll et al., 2005). The bamY gene contains an RpoN-dependent
promoter and the response regulator BamW is also an enhancer-binding
protein (Jurez et al., 2010). Addition of benzoate, p-cresol, or
p-hydroxybenzoate to cultures of E. coli heterologously expressing bamV
and bamW induced expression of a b-galactosidase gene fused to the bamY

promoter, demonstrating the role of this two-component system in
controlling aromatics metabolism (Jurez et al., 2010).
FgrM, which is a member of the enhancer-binding protein family as well
as a response regulator, is the master transcriptional regulator for flagellar
gene expression in Geobacter species (Ueki et al., 2011). FgrM interacts
with RpoN to control transcription of a number of flagella genes, including
the gene for FliA, which controls expression of some flagella-related genes,
including chemotaxis genes. Thus, it appears likely that the expression of
flagella-related genes is controlled in a cascade manner.
8.4. Chemotaxis
Unlike well-characterized motility systems such as those found in E. coli and

Bacillus subtilis, both of which have a single chemotaxis system, Geobacter
species contain multiple chemotaxis systems or homologs of the chemotaxis
system (Tran et al., 2008). One of the chemotaxis(-like) systems, designated
the a-group, is unique to Geobacter species and is predicted to be involved
in flagellar motility (Ueki et al., 2011). A second chemotaxis-like system,
designated the b-group and found in d-proteobacteria, is involved in the regu-
lation of the expression of extracellular proteins, such as the c-type
cytochromes, OmcS and OmcZ (Tran et al., 2011). Proteins of both the a
and b-groups were abundant in groundwater during acetate-stimulated in situ
uranium bioremediation (Wilkins et al., 2009).
Geobacter species appear to have an unusually large number of chemo-
receptor (MCP) genes (Aklujkar et al., 2009, 2010; Mth et al., 2003; Tran
et al., 2008). Several MCP genes are located in proximity to other chemo-
taxis genes on the genome in Geobacter species but most are scattered on
the genome. Some are predicted to play a role in chemotaxis (Ueki et al.,
2011). It is possible that other MCP genes are involved in signal transduc-
tion pathways mediated by chemotaxis-like systems.
8.5. Nucleotide-Based Second Messenger
Stringent response, originally observed during amino acid starvation in

E. coli, is affected by (p)ppGpp, which is known to act as a global regulator
in physiological adaptation to a variety of environmental changes
(Braeken et al., 2006; Potrykus and Cashel, 2008). In G. sulfurreducens,
ppGpp and ppGp were produced in response to nutrient limitations and
ppGpp accumulated as the result of oxygen exposure (DiDonato et al.,

2006). The production of ppGpp in G. sulfurreducens was dependent on
the rel gene encoding a homolog of the bifunctional RelA/SpoT protein,
which has both (p)ppGpp synthetase and hydrolase activity. Deleting rel
affected expression of genes involved in protein synthesis, stress responses,
and electron transport systems, and enhanced growth with fumarate as the
electron acceptor, but increased oxygen sensitivity and diminished the
capacity for Fe(III) reduction. Bioinformatic analysis suggested that genes
influenced by the Rel/ppGpp signaling system are also controlled by Fur
and RpoS (Krushkal et al., 2007).
Riboswitches, noncoding RNA elements found in the untranslated region
of mRNA, are known to sense and bind cellular metabolites to control gene
expression (Lioliou et al., 2010; Waters and Storz, 2009). Geobacter species
possess riboswitches termed GEMM (genes related to the environment,
membranes and motility) (Weinberg et al., 2007), which have been shown to
sense c-di-GMP in other bacteria (Sudarsan et al., 2008). G. uraniireducens
has the largest number of c-di-GMP riboswitch homologs among bacteria
whose genomes have been sequenced (Weinberg et al., 2007). In G.
sulfurreducens, genes known to be differentially regulated during metal reduc-
tion and electricity production, such as omcS and omcT, were found to contain
a c-di-GMP riboswitch signature in their noncoding region of mRNA
(Weinberg et al., 2007). The 50 untranslated region of omcS mRNA is critical
for omcS expression (B-C. Kim et al., unpublished data).
Another c-type cytochrome whose expression appears to be controlled
with a GEMM riboswitch is PgcA, which contains a GEMM riboswitch
sequence between the predicted RpoD-dependent promoter and the start
codon (Tremblay et al., 2011b). An increase in c-di-GMP in E. coli results
in the upregulation of lacZ under the control of the pgcA-associated GEMM
riboswitch (B-C. Kim et al., unpublished data). Adaptive evolution of G.
sulfurreducens for improved growth on Fe(III) oxide selected for strains that
had either a single base-pair change or a one-nucleotide insertion in the
GEMM riboswitch of the pgcA gene. Introduction of either of the GEMM
riboswitch mutations in the pgcA gene into the wild-type strain increased
the abundance of pgcA transcripts, consistent with increased expression of
pgcA in the adapted strains.
8.6. Summary Statement on Regulation
The abundance of regulatory genes and novel sensing capabilities found in

Geobacter species suggest that they are highly attuned to their
environment and have evolved to be able to sense a wide diversity of envi-

ronmental cues. For the most part, even the regulatory systems that have
already been studied so far have only been examined in a rather prelimi-
nary manner and there are many other regulatory systems that have yet
to be investigated. A better understanding of these systems will greatly
aid the development of models to predict the activity of Geobacter species
under different environment conditions.
9. ENVIRONMENTAL SYSTEMS BIOLOGY OF GEOBACTER
The availability of pure cultures of Geobacter species closely related to

those that are abundant in Fe(III)-reducing environments has made it
possible to take a systems approach to the study of Geobacter ecology in
subsurface environments. For example, quantifying key gene transcripts
or proteins can provide a diagnosis of the in situ physiological status of
Geobacter species, providing insights into metabolic patterns that are likely
to be much different than when the microorganisms were grown under
nutritionally replete conditions in the laboratory. Even estimating how fast
microorganisms are metabolizing in natural environments can be difficult,
especially in subsurface environments which are difficult to sample.
Understanding in situ physiological status is key for bioremediation,
making it possible to rationally design strategies to modify the in situ
activity of Geobacter species (Lovley, 2003; Lovley et al., 2008).
9.1. Environmental Transcriptomics and Proteomics
Several strategies were investigated to elucidate the rate of activity of Geo-

bacter species in the subsurface. One successful approach was based on
monitoring gene transcript abundance (Holmes et al., 2005; Williams
et al., 2011) or protein abundance (Wilkins et al., 2011; Yun et al., 2011b)
of the key TCA cycle enzyme citrate synthase. The citrate synthase
sequences of Geobacter species are more closely aligned with those of
eukaryotes, rather than other prokaryotes (Bond et al., 2005; Mth
et al., 2003), simplifying the task of designing PCR primers to specifically
amplify citrate synthase sequences of Geobacter species. Chemostat studies
with G. sulfurreducens demonstrated a direct correlation between rates of
acetate metabolism and transcript abundance for citrate synthase (Holmes
et al., 2005). Subsequent field studies in which the in situ levels of
Geobacter citrate synthase gene transcripts were monitored demonstrated

that as acetate availability in the groundwater was artificially manipulated,
the metabolism of the in situ Geobacter community responded accordingly,
increasing expression of citrate synthase when acetate concentrations were
elevated and repressing expression when acetate levels dropped (Holmes
et al., 2005; Williams et al., 2011). A similar metabolic response was noted
when citrate synthase protein levels were quantified with an antibody-
based approach (Yun et al., 2011b).
Attempts to monitor bulk rates of respiration by quantifying the tran-
script abundance of genes specifically involved in electron transfer pro-
cesses was less successful (Chin et al., 2004), but recent studies with
sulfate reducers have demonstrated that important insights into per-cell
rates of metabolism might be obtained with such an approach (Miletto
et al., 2011; Villanueva et al., 2008), suggesting that it might be productive
to revisit this approach in Geobacter species.
Recent studies demonstrated that the abundance of a predominant
c-type cytochrome in groundwater correlated well with the activity of Geo-
bacter species and the effectiveness of uranium bioremediation (Yun et al.,
2011b). Subsequent functional analysis suggested that this cytochrome
might function similarly to the OmcS of G. sulfurreducens.
As important as it is to understand rates of metabolism, it is equally
important to understand the factors that control those rates. Quantifying
key gene transcripts or proteins has been shown to be a useful tool for
diagnosing which nutrients or stresses might be limiting the growth of the
subsurface Geobacter community. For example, measuring transcript abun-
dance (Holmes et al., 2004c; Mouser et al., 2009b) or protein abundance
(Yun et al., 2011b) of the nitrogen-fixation protein NifD can indicate
whether Geobacter species in the subsurface are limited for ammonium
and need to fix atmospheric nitrogen. This information can guide bioreme-
diation because it may be beneficial for cells to be ammonium-limited
during uranium bioremediation, but for bioremediation of hydrocarbon-
contaminated groundwater ammonium limitation is likely to slow contami-
nant removal.
In a similar manner, molecular analysis of the in situ physiological status
of the subsurface Geobacter community has provided insights into phos-
phate limitation (N'Guessan et al., 2009), acetate availability (Elifantz
et al., 2011), iron limitation (O'Neil et al., 2008), and oxidative stress during
uranium bioremediation (Mouser et al., 2009a). Monitoring transcript
abundance for a ribosomal protein made it feasible to estimate growth
rates of Geobacter species during uranium bioremediation and is expected
to be an important tool in evaluating the proposed slow growth of
microorganisms in undisturbed subsurface environments (Holmes et al.,

2011a). The increased expression of a key Geobacter enzyme in the degrada-
tion of aromatic hydrocarbons in response to hydrocarbon contamination in
groundwater was documented with an antibody-based approach (Yun et al.,
2011b). Continued analysis of Geobacter communities with high-throughput
proteomics (Callister et al., 2010; Wilkins et al., 2009) and broad transcriptomic
approaches (Holmes et al., 2009) is expected to identify other key gene tran-
scripts and proteins that will serve as diagnostic tools for better understanding
the ecology of Geobacter species during bioremediation and in undisturbed
soils and sediments.
9.2. BUGS (Bottom-Up Genome-Scale) Modeling
A major goal in microbial ecology is to be able to not only describe the

distribution of microorganisms and their activity, but to predict microbial
distributions and interactions with other microorganisms and the environ-
ment under a diversity of environmental conditions. One strategy for this
is to couple genome-scale metabolic models with the appropriate models
that can describe physical/chemical conditions and their changes in
response to predicted microbial activity (Lovley, 2003; Lovley et al., 2008;
Mahadevan et al., 2011; Zhao et al., 2010).
We have termed this modeling approach bottom-up genome-scale
modeling, abbreviated BUGS modeling, to differentiate it from the
increasingly popular top-down approach of beginning with a global analy-
sis of genes, gene transcripts, and proteins in environments of interest. The
two approaches are complementary. An advantage of the top-down com-
munity wide approach is that it can rapidly provide a parts list, an
accounting of the diversity of genes and proteins and their relative abun-
dance. However, this is a highly descriptive approach and it is difficult to
make predictions about the response of microorganisms to changes in envi-
ronmental conditions from such lists. In BUGS modeling, genome-scale
metabolic models are made for the microorganisms that predominate in
an environment of interest and their interaction with each other and their
environment is modeled. BUGS modeling is a slower, iterative process, but
in the end provides a knowledge base, based on first principles of microbial
physiology, that should have broad applicability and predictive power.
Subsurface environments in which Geobacter species predominate have
proven to be good test cases for the BUGS modeling concept. The addition
of acetate to groundwater to stimulate dissimilatory metal reduction results
in a bloom of Geobacter species, which are the primary microorganisms
influencing subsurface biogeochemistry during this period. Further, Geo-

bacter is one of the rare examples where isolates of the species that pre-
dominate in the environment of interest are available in pure culture. In
these still early days of the annotation of microbial genomes, the study of
relevant pure cultures is necessary because many important physiological
features, as well as the function of many genes, cannot be ascertained
solely from genomic sequences.
The details of the generation of genome-scale models for environmental
studies have recently been reviewed (Mahadevan et al., 2011) and will not
be repeated here. The initial genome-scale modeling of G. sulfurreducens
proved to be an important driver for hypothesis-driven research and rev-
ealed important metabolic features (Mahadevan et al., 2006, 2011; Yang
et al., 2010). For example, the mechanisms for acetate uptake, catabolic
and anabolic utilization, as well as energy conservation during reduction
of internal (fumarate) or external (Fe(III)) electron acceptors were
elucidated in an iterative process of laboratory experimentation and in sil-
ico modeling. The genome-scale model of G. sulfurreducens was an impor-
tant tool for analyzing the incorporation of carbon into amino acids, and
revealed that isoleucine was synthesized via the citramalate pathway
(Risso et al., 2008b). Continued development of genome-scale metabolic
models in other Geobacter species (Sun et al., 2009) and closely related
organisms (Sun et al., 2010) is identifying commonalities in metabolic
strategies as well as adding new metabolic modules, such as the pathways
for the degradation of aromatic compounds found in G. metallireducens
and other Geobacter species.
To date, BUGS modeling of the biogeochemical impacts of Geobacter
species has been applied to relatively simple subsurface environments.
In initial studies, the genome-scale metabolic model of G. sulfurreducens
was coupled with reactive transport models to determine geochemical
changes when acetate was added to groundwater to stimulate in situ ura-
nium bioremediation (Scheibe et al., 2009). Initial results were encouraging.
The predominance of Geobacter species during acetate-amended ura-
nium bioremediation is rare and some of the most important ecological
interactions in soils and sediments are those between different
microorganisms. Therefore, in order to expand the BUGS modeling con-
cept, the interactions between Rhodoferax and Geobacter species in the
subsurface were modeled. Like Geobacter species, Rhodoferax fer-
rireducens is an acetate-oxidizing Fe(III) reducer (Finneran et al., 2003).
Rhodoferax has a higher growth yield from acetate, but slower growth rate
than Geobacter and Rhodoferax cannot fix nitrogen whereas Geobacter
can. First, a genome-scale model of R. ferrireducens was generated (Risso
et al., 2009). Then the genome-scale models of G. sulfurreducens and R.

ferrireducens were used to model their growth under different environmen-
tal conditions (Zhuang et al., 2010). The modeling predicted that, as has
been observed experimentally (Mouser et al., 2009b), Geobacter and
Rhodoferax species are likely to coexist in subsurface environments in
which the slow degradation of organic matter deposited with the sediments
drives microbial metabolism. Where ammonium concentrations are rela-
tively high, Rhodoferax will predominate. However, the modeling pre-
dicted that when acetate is added to the groundwater to promote the
growth of Fe(III) reducers, Geobacter outgrows Rhodoferax because of
its much faster growth rate. This prediction is consistent with what is
observed during bioremediation. The BUGS modeling approach is now
being expanded to evaluate the competition between Geobacter species
and sulfate-reducing Desulfobacter species, which compete with Geobacter
species for acetate. Over time more complex communities, potentially first
grown in the laboratory (Miller et al., 2010) and then studied in the field,
can be modeled.
At some point, it may be possible to greatly accelerate the BUGS
modeling process by building models from genomes sequenced directly
from the environment. However, as of now, gene annotation and the abil-
ity to predict even simple physiological properties, such as growth rate,
from the genome sequence are not sufficiently advanced.
10. BIOGEOCHEMICAL IMPACTS OF GEOBACTER SPECIES
Previous reviews have detailed many of the substantial geochemical

impacts that Geobacter species can have on anaerobic soils and sediments
(Lovley, 1991, 1993, 1995, 2000a,b), and these topics will not be covered in
detail here. Important geochemical changes that take place in Fe(III)- and
Mn(IV)-reducing environments in which Geobacter species are abundant
can include the production of magnetite, siderite, and other Fe(II) and
Mn(II) minerals; the release of iron, trace metals, metalloids, and phos-
phate into pore waters; other changes that influence the pH and ionic
strength of pore waters; and changes in soil porosity as the result of reduc-
tion of Fe(III) in clays. The degradation of organic carbon in soils and
sediments coupled to the reduction of Fe(III) and Mn(IV) can contribute
significantly to anaerobic organic matter degradation with the release of
carbon dioxide. Any organic matter degraded in this manner results in less
reduction of sulfate and less production of methane. Ions of metals and
metalloids are natural constituents of soils and sediments, and as noted in

other sections, the ability of Geobacter species to reduce these can influ-
ence their geochemical fate.
In some instances, the activity of Geobacter species in subsurface
environments can have deleterious impacts on groundwater quality. For
example, undesirably high concentrations of Fe(II) and Mn(II) as the
result of microbial reduction of Fe(III) and Mn(IV) are common
(Anderson and Lovley, 1997; Chapelle and Lovley, 1992; Lovley, 1997).
Geobacteraceae are abundant in groundwaters with high arsenic con-
centrations in which Fe(III) reduction has been implicated in the release
of arsenic (Hry et al., 2010; Islam et al., 2004a; Smedley and Kinniburgh,
2002; Weldon and MacRae, 2006). Possibilities for arsenic fluxes from
sediments to groundwater include release of arsenic adsorbed onto Fe(III)
oxide and/or the reduction of As(V) to As(III), which is more soluble.
For example, Geobacter species are thought to play a key role in the mobili-
zation of arsenic from West Bengal sediments (Hry et al., 2008; Islam et al.,
2004a,b, 2005a,b) where arsenic release takes place after Fe(III) reduction,
rather than occurring simultaneously (Islam et al., 2004b). It has been
proposed that Geobacter species related to G. uraniireducens and G. lovleyi
may be the primary catalysts for As(V) reduction (Hry et al., 2010) but
As(V) reduction in these species has not yet been documented.
Some Geobacter species can methylate mercury and their activity may
be an important source of this environmental toxin in iron-rich freshwater
sediments (Fleming et al., 2006). Pure cultures capable of mercury methyl-
ation include Geobacter strain CLFeRB (Fleming et al., 2006) as well as
G. hydrogenophilus, G. metallireducens, and G. sulfurreducens and the
closely related Desulfuromonas palmitatis (Kerin et al., 2006). Environ-
mental conditions that control the extent to which Geobacter species
methylate mercury are beginning to be examined (Schaefer and Morel,
2009; Schaefer et al., 2011) and warrant further study.
11. PRACTICAL APPLICATIONS OF GEOBACTER SPECIES
11.1. Bioremediation: Natural Attenuation and Engineered
11.1.1. Aromatic Hydrocarbons
Geobacter species are often important components of the microbial commu-

nity in aquifers polluted with petroleum or landfill leachate (Alfreider and
Vogt, 2007; Botton et al., 2007; Holmes et al., 2007; Lin et al., 2005, 2007;
Rling et al., 2001; Rooney-Varga et al., 1999; Staats et al., 2011; Van
Stempvoort et al., 2009; Winderl et al., 2007, 2008) which can be attributed,
at least in part, to the ability described above of some Geobacter species to
degrade aromatic compounds. Prior to contamination most shallow aquifers
are aerobic, but anaerobic conditions rapidly develop once organic con-
taminants are introduced. Fe(III) is generally an abundant electron acceptor
for anaerobic degradation and early studies demonstrated a removal of
aromatic hydrocarbon contaminants from petroleum-contaminated ground-
water associated with geochemical signatures for Fe(III) reduction (Lovley
et al., 1989). Subsequent analysis demonstrated an abundance of Geobacter
species in the Fe(III) reduction zone (Anderson et al., 1998; Holmes et al.,
2004c, 2007; Lovley et al., 1989; Nevin et al., 2005; Rooney-Varga et al.,
1999), accounting for 41% of the active microbial community in the ground-
water (Holmes et al., 2007). In a similar manner, 25% of the microbial
community comprised Geobacter species in a landfill leachate-contaminated
aquifer (Rling et al., 2001). Quantifying specific genes or proteins known to
be involved in the degradation of aromatic compounds has further demon-
strated the importance of Geobacter species in naturally removing aromatic
contaminants (Hosoda et al., 2005; Kane et al., 2002; Kuntze et al., 2008, 2011;
Winderl et al., 2007, 2008; Yun et al., 2011b).
The finding that Geobacter species could reduce chelated Fe(III) faster
than Fe(III) oxides (Lovley and Phillips, 1988a; Lovley and Woodward,
1996) and that electron shuttles promoted Geobacter reduction of Fe(III)
oxide (Lovley et al., 1996a) led to studies evaluating whether the addition
of Fe(III) chelators could stimulate the degradation of aromatic
hydrocarbons (Lovley et al., 1994, 1996b). In the presence of these
stimulants, even benzene could be degraded as rapidly with Fe(III) as
the electron acceptor as it could with the introduction of oxygen.
The most practical method for enhancing electron-acceptor availability
to Geobacter species involved in the degradation of organic contaminants
in contaminated groundwater or aquatic sediments may be the concept
of subsurface snorkels (Lovley, 2011c). Studies with G. metallireducens,
as well as natural communities, demonstrated that providing an electrode
as an electron acceptor may be a good strategy for stimulating the degrada-
tion of aromatic hydrocarbon contaminants (Zhang et al., 2010). Graphite
electrodes may be the best option as they are inexpensive and durable and
have the added advantage of adsorbing aromatic contaminants on their
surface. This colocalizes the contaminant, the electron acceptor, and
the Geobacter species at the electrode surface. Initial studies focused on
the use of relatively complex systems in which the potential of the
electrode was electronically poised (Zhang et al., 2010). However, it may

be sufficient to insert conductive rods into contaminated sediments with
a portion of the conductive rod extending into aerobic soil, water, or the
atmosphere. With such subsurface snorkels, the portion of the rod in the
anaerobic soil can function as an electron acceptor to promote the growth
of Geobacter capable of degrading the contaminants while the portion of
the rod in the aerobic environment functions as a cathode (Lovley, 2011c).
11.1.2. Uranium and Related Metals and Metalloids
The ability of Geobacter to reduce soluble ions of metals to less soluble forms
shows promise as a bioremediation tool. Metals may be removed from water
in this manner in reactors, or stimulating the activity of Geobacter species for
in situ immobilization is an option. In some instances, Geobacter species
might naturally attenuate the movement of metals via reduction.
Uranium has been the contaminant metal of greatest focus because the
rapid kinetics of bacterial U(VI) reduction and low solubility of U(IV)
make this process an attractive option for removing uranium from
groundwaters below drinking water standards (Williams et al., 2011, and
references therein). The rather nonspecific nature in which Geobacter spe-
cies reduce U(VI) (see above) and the fact that even in uranium-con-
taminated environments U(VI) is likely to be a minor electron acceptor
(Finneran et al., 2002a) make it difficult to definitely determine if
Geobacter species are the agents for U(VI) reduction in studies in which
dissimilatory metal reduction has been stimulated to promote uranium bio-
remediation. However, the consistent pattern of effective U(VI) removal
being associated with increased growth and activity of Geobacter species
at least at some sites (Williams et al., 2011, and references therein) suggests
that Geobacter species play a role.
Stimulating the activity of Geobacter species may also remove a variety
of other toxic metals that Geobacter species have the potential to reduce
in pure culture, but the reduction of these contaminants may be indirect
in subsurface environments, because as noted above in Section 5, these
electron acceptors can also be reduced by Fe(II) that Geobacter species
generate during Fe(III) oxide reduction.
Although the commonly considered approach to stimulating the activity
of Geobacter species for bioremediation of uranium and related con-
taminants is to add organic electron donors, a more effective approach
might be to provide Geobacter species electrons with electrodes (Gregory
and Lovley, 2005). Long-term stimulation of anaerobic respiration has
several potential negative impacts (Williams et al., 2011). These include

(1) release of trace metals and arsenic that were associated with Fe(III)
oxides into the groundwater (Burkhardt et al., 2010), (2) deterioration of
the groundwater quality from accumulations of dissolved Fe(II) or sulfide,
and (3) aquifer plugging due to biomass or mineral accumulations
(Williams et al., 2011). Further, reductive immobilization of uranium in this
manner leaves the uranium contamination in the subsurface.
Therefore, a better alternative may be to feed Geobacter species
electrons with electrodes (Gregory and Lovley, 2005). Maintenance of
the electron addition to the subsurface with electrodes is much simpler
than complex pumping strategies for the controlled introduction of organic
electron donors and the electrode strategy is sustainable, easily powered
with solar panels. Further, this strategy specifically provides electrons for
the reduction of the soluble contaminant of interest and the U(IV) pro-
duced precipitates on electrodes. It would be a simple matter to periodi-
cally remove the electrodes, extract the U(IV) under aerobic conditions
in bicarbonate (Phillips et al., 1995), and return the electrodes to the
subsurface. This approach would alleviate all the negative side effects of
adding the organic electron donors listed above as well as remove the
uranium from the subsurface.
11.1.3. Chlorinated Contaminants
In a similar manner, providing electrons to Geobacter species in the subsur-

face with electrodes may be an effective strategy for stimulating reductive
dechlorination (Strycharz et al., 2008, 2010). Although no Geobacter spe-
cies are known to completely dechlorinate chlorinated solvents, electrodes
have the potential to specifically localize the electron donor and the
dechlorinating organisms. Therefore, cathodes colonized with Geobacter
species and positioned near source zones of chlorinated solvents could con-
vert the solvents to the much more soluble products, susceptible to aerobic
degradation, that could be degraded downgradient at the anode which
produces oxygen (Lovley and Nevin, 2011). As noted above, Geobacter
species have frequently been detected in subsurface environments con-
taminated with chlorinated solvents and in dechlorinating enrichments.
The formation of reactive Fe(II) minerals by Geobacter species during
Fe(III) oxide reduction may accelerate the removal of carbon tetrachloride
(McCormick et al., 2002). In a similar manner, humic substances may
provide an electron shuttle to promote carbon tetrachloride reduction
(Cervantes et al., 2004).
11.2. Producing Methane from Organic Wastes and

Hydrocarbon Deposits
Conversion of organic wastes and biomass to methane has been a long-

standing bioenergy strategy whose use could be expanded if the process
could be accelerated and made more consistently stable. Geobacter species
may have a role in this process development because the ability of Geo-
bacter species to function as syntrophs may permit them to significantly
contribute to rapid conversion of organic matter to methane.
Molecular analyses have demonstrated that Geobacter and closely related
species can account for over 20% of the microbial community in the
methanogenic aggregates that form in anaerobic digesters treating brewery
wastes (Morita et al., 2011; Werner et al., 2011). Detailed analysis of
aggregates from one of these digesters demonstrated that the aggregates
had a conductivity similar to that of Geobacter co-culture aggregates (Morita
et al., 2011). The temperature dependence of the conductance suggested an
organic metallic-like conductivity, similar to that observed (Malvankar et al.,
2011b) in pilin preparations of G. sulfurreducens and G. sulfurreducens bio-
films. Several lines of evidence suggest that methanogenic microorganisms
might be able to directly accept electrons and that direct electron transfer,
rather than interspecies hydrogen or formate transfer, was the primary mech-
anism for electron exchange with the aggregates (Morita et al., 2011). The
understanding that Geobacter species, and possibly other microorganisms,
may be directly transferring electrons to methanogens, via direct interspecies
electron transfer, may lead to new reactor designs to better promote this
interaction and accelerate the process (Lovley, 2011a,c).
It has also been proposed that the capacity of Geobacter species to func-
tion in methanogenic syntrophic interactions can be used to enhance the
recovery of hydrocarbons from coal and hydrocarbon deposits (Jones et al.,
2010; Siegert et al., 2010). In fact, in an enrichment culture converting coal
to methane, the most important microorganisms appeared to be Geobacter
and Methanosaeta species (Jones et al., 2010), which is similar to what was
found in wastewater methanogenic aggregates (Morita et al., 2011). This
suggests that principles for Geobacter contributions to methanogenic waste-
water treatment may apply to hydrocarbon recovery from the subsurface.
11.3. Microbial Fuel Cells, Electrosynthesis, and

Bioelectronics
There is significant interest in the development of large-scale microbial

fuel cell systems for wastewater treatment. Given the consistent
enrichment of Geobacteraceae on anodes of effectively operating microbial

fuel cells, pre-enrichment of anodes with Geobacter species may be an
important step in scale-up (Cusick et al., 2011).
There may be significant potential for increasing the current output of
microbial fuel cells via strain selection/design (Izallalen et al., 2008; Yi
et al., 2009). The anode of a microbial fuel cell is not a natural electron
acceptor, and thus it is unlikely that there has been significant selective
pressure on Geobacter species to optimize current production under the
conditions found in microbial fuel cells (Lovley, 2006a). For example,
increasing pilin expression of G. sulfurreducens, via strain selection or
genetic engineering, increased biofilm conductivity and current production
(Malvankar et al., 2011b). As more is learned about the mechanisms for
electron transfer to electrodes in Geobacter species, it may be possible to
further enhance power output.
Even without strain improvement there may be some short-term practical
applications for microbial fuel cells, such as powering electrical devices in
remote locations, such as at the bottom of the ocean (Tender et al., 2008).
The fact that Geobacter species are often the primary microorganisms col-
onizing electrodes harvesting current from a diversity of environments
suggests that they are likely to play an important role in any applications
of microbial fuel cells in which current is harvested in open environments
in which there will be competition for anode colonization. Geobacter-based
sensors may also be practical (Davila et al., 2010). Novel system designs
make it feasible to consider producing current with Geobacter species, even
in completely aerobic environments (Nevin et al., 2011b). Electrodes
deployed in subsurface environments are naturally colonized by Geobacter
species (Williams et al., 2010) and may function as sensors of subsurface
microbial activity (Tront et al., 2008; Williams et al., 2010).
Microbial electrosynthesis is a process in which electrons are provided to
microorganisms colonizing an electrode to support the reduction of carbon
dioxide to organic compounds that are excreted from the cells (Lovley,
2011b; Lovley and Nevin, 2011; Nevin et al., 2010, 2011a). When powered
with solar technology, microbial electrosynthesis is an artificial form of
photosynthesis in which sunlight drives the conversion of carbon dioxide
and water to organic compounds and oxygen. Proof-of-concept studies
have demonstrated acetate production with acetogenic microorganisms as
the catalysts (Nevin et al., 2010, 2011a).
Genome annotation led to the surprising discovery of enzymes for car-
bon dioxide fixation in some Geobacteraceae (Aklujkar et al., 2010). Within
the G. metallireducens genome, a pair of genes is predicted to encode an
ATP-dependent citrate lyase, which would allow the reverse TCA cycle
to produce acetyl-CoA. Further, genes for all of the identified enzymes of

the dicarboxylate/4-hydroxybutyrate cycle of carbon dioxide fixation
are predicted in the G. metallireducens genome. G. metallireducens is
also capable of electrosynthesis, and investigations with genetically
modified strains of other Geobacter species are ongoing because of the
ability of Geobacter species to interact so effectively with electrodes.
G. sulfurreducens can also use electrons derived from an electrode to
reduce protons to hydrogen (Geelhoed and Stams, 2011), potentially
providing a renewable catalyst that is much less expensive than the metal
catalysts typically employed for hydrogen production.
One of the most exciting practical applications for Geobacter species
could be bioelectronics. Electronically functional biomaterials are very
attractive because they can be synthesized from relatively inexpensive
feedstocks and do not contain toxic components (Hauser and Zhang,
2010). Further, conductive materials comprising living bacteria are self-
renewing because bacteria can self-repair and replicate. Initial studies have
already demonstrated the possibility of tuning the electronic properties of
Geobacter biofilms via simple genetic engineering and more sophisticated
modifications are feasible. Further elucidation of the mechanisms for elec-
tron transport along pili and ability of cytochromes to function as capacitors
could aid in the biomimetic design of new materials. Therefore, it is
expected that microbially based electronically functional materials will have
significant potential for next-generation biotechnological applications.
12. CONCLUSIONS
Studies to date have demonstrated the importance of Geobacter species to

the anaerobic degradation of organic matter in sedimentary environments
and its importance in iron, manganese, and trace-metal biogeochemistry.
Geobacter species can naturally attenuate the migration of organic and
metal contaminants, and strategies for artificially stimulating contaminant
removal by Geobacter species are being developed.
The novel electrical properties of Geobacter species, and their pili and
cytochromes, coupled with their ability to form direct electrical connections
with man-made electronics, are amazing and provide new paradigms for the
function of microbial communities and the development of next-generation
bioelectronics. It has been suggested that if it were not for the bacterium
GS-15, we would not have radio and television today because one of the
first discovered properties of G. metallireducens was its ability to make
magnetite and it was the study of magnetite lodestone that contributed to the
early understanding of electricity (Verschuur, 1993). Therefore, it may be
fitting that one of the most recently discovered properties of Geobacter
species, metallic-like conductivity along pili, has the possibility to make a
more direct contribution to further the development of electronics.
Our understanding of the ecology, physiology, biochemistry, and
bioelectronics of Geobacter is very rudimentary. Rapid advancements in
omic technologies greatly facilitated a substantial increase in the under-
standing of Geobacter over the past decade. Continued functional genomic
analyses of many aspects of Geobacter metabolism and genetic regulation
will be essential for continued progress. Further, contributions from other
fields, such as physics, materials science, and engineering, will be important
not only to increase basic understanding of Geobacter species but also for
the development of many promising, novel applications.
ACKNOWLEDGMENTS
Research on Geobacter species in our laboratory is currently funded by (1)

the Office of Science (BER) U.S. Department of Energy through Cooper-
ative Agreement No. DE-FC02-02ER63446, Award No. DE-SC0004114,
Award No. DE-SC0004080, Award No. DE-SC0004814, Award No. DE-
SC0004485, and Award No. DE-SC0006790; (2) the Advanced Research
Projects Agency-Energy (ARPA-E), U.S. Department of Energy, under
Award No. DE-AR0000087 and Award No. DE-AR0000159; and (3) the
Office of Naval Research Grant No. N00014-09-1-0190, Grant No.
N00014-10-1-0084, and Grant No. N00014-10-C-0184.
We thank Anna Palmissano and Dan Drell of the Department of
Energy and Linda Chrissey of the Office of Naval Research for
their invaluable insights, helpful discussions, and long-term support of
Geobacter research.
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Geobacter: The Microbe Electric's Physiology, Ecology, and Practical Applications

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Geobacter: The Microbe Electrics

Physiology, Ecology, and Practical

Department of Microbiology and Environmental Biotechnology Center, University of

Geobacter species specialize in making electrical contacts with extracellular

ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 59 Copyright # 2011 by Elsevier Ltd.

appears to be an important process in anaerobic wastewater digesters.

7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide . . . . . . . 34

Geobacter species represent a rare example of a genus of microorganisms

magnetite from microbial Fe(III) reduction, (4) anaerobic oxidation of an

Dominance in Current Metallic-like

1985 1990 1995 2000 2005 2010 2015

U(VI) Humics as Electrode-

Figure 1 Time line of important discoveries associated with Geobacter species.

Figure 2 Publications and citations each year with Geobacter as a topic

2. DISTRIBUTION AND ABUNDANCE OF GEOBACTER

The hallmark physiological capability of Geobacter species is their ability

Genome Electron donors Other Optimal

Genome Electron donors Other Optimal

Geobacter Contaminated Ac, Buty, Bzo, PCIO, Fe AQDS, 30 Coates et al.

Genome Electron donors Other Optimal

Geobacter Acetate- Ac, Act, Bzo, PCIO, 2530 Holmes

Figure 3 Neighbor-joining tree showing the phylogenetic relationship within

3. BRIEF DESCRIPTION OF GEOBACTER SPECIES

A significant number of pure culture isolates of Geobacter species are

important physiological differences. In addition to producing more current

G. plymouthensis. Some of these isolates were recovered from surface

4. PHYLOGENY AND GENOMIC RESOURCES

Geobacter species are in the family Geobacteraceae, which is within the

Geobacter species can use a diversity of electron acceptors to support anaer-

At the circumneutral pH in most environments in which Geobacter thrive, Fe

After Fe(III) reduction, electron transfer to electrodes is probably the

5.3. Other Extracellular Electron Acceptors

Fe(III)-reducing microorganisms reduced Au(III), the Geobacter species

5.4. Other MicroorganismsSyntrophy

In the absence of alternative electron acceptors, Geobacter species can

6.1. Acetate, Other Fatty Acids, Hydrogen, Electrodes,

The universal ability of all Geobacter species to oxidize acetate with Fe

NAD-dependent ferredoxin:NADP oxidoreductase (Nfn) complex, and all

for electron transfer from electrodes to Geobacter species may be different

6.2. Aromatic Compounds

As detailed later, Geobacter species are often abundant within zones of

Figure 5 Aromatic hydrocarbon oxidation pathways in Geobacter species.

barrier associated with ring reduction, class II benzoyl-CoA reductases

Additional reactions (Fig. 5) complete the conversion of benzoyl-CoA to 3-

cyclohexadienoyl-CoA hydratase, the hydroxyenoyl-CoA dehydrogenase,

7. EXTRACELLULAR ELECTRON TRANSFER

Effective extracellular electron transfer is one of the hallmark physiologi-

species is accomplished through unique mechanisms that have yet to be

7.1. Microbial Nanowires

One of the most surprising discoveries in the study of extracellular electron

was responsible for the electron transfer. Studies on the impact of pH

7.2. Cytochromes and Multicopper Proteins

One of the most striking features of Geobacter species is their abundant

but its periplasmic location makes direct reduction of Fe(III) unlikely

OmcB is encoded downstream from another cytochrome, designated

to date, only OmcZ is absolutely necessary for high-density current pro-

has different expression patterns than OmpB (Holmes et al., 2008).

7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide

The role of other outer-surface cytochromes in Fe(III) oxide reduction is

the terminal steps in electron transfer to Fe(III) and other extracellular

7.4. Model for Extracellular Electron Transfer to Electrodes

Like Fe(III) oxide, electrodes represent an insoluble, extracellular electron

aeruginosa were not conductive. Evaluation of strains of G. sulfurreducens