Geobiology (2008), 6, 225– 231 DOI: 10.1111/j.1472-4669.2008.00148.

Extracellular electron transfer: wires, capacitors, iron lungs,

O RIGINA L electron
Extracellular
Blackwell A R T I CLtd
Publishing Ltransfer
ES

and more
DE R E K R . LOVL E Y

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA

Received 7 October 2007; accepted 25 February 2008

Corresponding author: Derek R. Lovley. Tel: 413-545-9651; fax: 413-545-1598; e-mail: [email protected].

‘and by the help of microscopes, there is nothing so small, as to cycles in sedimentary environments, and also influences the
escape our inquiry; hence there is a new visible world discovered fate of a diversity of trace metals and phosphate (Lovley, 1993;
to the understanding .’ Thamdrup, 2000). Anaerobic oxidation of organic contaminants
Robert Hooke with the reduction of Fe(III) is important in groundwater
bioremediation (Lovley et al., 1989; Lovley, 1997), and stim-
Terry Beveridge’s broad and insightful studies of microbe– ulating dissimilatory metal reduction has shown promise as a
metal interactions, powered in large part by amazing microscopy, method for immobilizing toxic metals in the subsurface
have served as an inspiration and provided guidance for many (Anderson et al., 2003; Lloyd et al., 2003). Oxidation of
of us working in this field. This perspective deals with just one organic matter coupled to electron transfer to electrodes is a
of the aspects of microbe–metal interactions to which Terry potential strategy for harvesting electricity from the environ-
significantly contributed, extracellular electron transfer. ment and waste organic matter (Lovley, 2006a).
For purposes of this perspective extracellular electron transfer The primary goal for many investigators of extracellular
is defined as the process in which electrons derived from the electron transfer is to determine how electrons are transferred
oxidation of electron donors are transferred to the outer surface to the electron acceptors and the factors controlling the rate
of the cell to reduce an extracellular terminal electron acceptor. and extent of this process. With this information in hand, it
Extracellular electron acceptors may be soluble, yet reduced may be possible to optimize practical applications and better
outside the cell because they are too large to enter the cell, as model natural processes. Some of the potentially important
is probably the case for humic substances (Lovley et al., 1996, questions still remaining in extracellular electron transfer are
1998). Some soluble metal species, such as U(VI) complexed discussed below in relation to key publications of Terry’s that
with carbonate, or Fe(III) chelated with citrate, are often reduced have led to the development of these questions and/or have
extracellularly (Lovley et al., 1991; Marshall et al., 2006; provided the techniques that may lead to their answers.
Coppi et al., 2007; Gralnick & Newman, 2007; Shelobolina
et al., 2007) as is dimethyl sulfoxide (Gralnick et al., 2006).
WHAT CONSTITUTES A ‘MICROBIAL
It has also been proposed that organisms involved in the
NANOWIRE’ AND ARE THEY THE FINAL
syntrophic degradation of organic compounds may make the
CONDUIT FOR ELECTRON TRANSFER TO
appropriate connections to directly exchange electrons (Stams
EXTRACELLULAR ELECTRON ACCEPTORS
et al., 2006). However, extracellular electron transfer to
SUCH AS FE(III) OXIDES AND ELECTRODES?
minerals, such as Fe(III) and Mn(IV) oxides (Lovley, 1991;
Lovley et al., 2004; Gralnick & Newman, 2007; Shi et al., Terry was involved in some of the earliest work on the concept
2007) and to electrodes (Lovley, 2006a,b), is probably of the that some structures emanating from microorganisms may be
greatest interest at present because of their environmental conductive and might serve as a conduit for extracellular electron
significance and practical applications. Oxidation of organic transfer (Gorby et al., 2006). ‘Microbial nanowires’ (Reguera
matter coupled to the reduction of Fe(III) and Mn(IV) oxides et al., 2005, 2006; Gorby et al., 2006) are certainly an attractive,
plays an important role in the carbon, iron, and manganese easily visualized, explanation for extracellular electron transfer.

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226 D. R. LOVLEY

However, some caution in this area is probably warranted. ductivity in Geobacter nanowires by laying the filaments across
It is not uncommon these days for some investigators to interdigitated electrodes and querying conductive properties.
confer ‘nanowire’ status on just about any filament emanating To date, such studies have proven to be technically challenging
from microorganisms, even those not known to be capable of and difficult to interpret.
extracellular electron transfer, often without even examining Even after the nature of conductivity of the nanowires is
the filaments to determine if they are, in fact, electrically elucidated, the challenge still remains to definitely determine
conductive. whether these conductive structures are the final conduit to
Even for the few instances in which electrically conductive Fe(III) oxides and electrodes. It is tempting to suggest that
filaments have been documented (Reguera et al., 2005; Gorby they are because they could have the potential to greatly
et al., 2006), much more investigation is required before it extend the electronic reach of the organisms. However, to
can be definitively stated that electrons actually flow from date the evidence is rather circumstantial and it has been
microorganisms to extracellular electron acceptors along these previously noted (Gorby et al., 2006) that ‘additional research
structures. It is important to recognize that such electron designed to critically and comprehensively evaluate far-field
transfer has never been directly demonstrated, only inferred electron transfer via nanowires is needed to confirm these
from circumstantial evidence. More information is required intriguing observations’. In G. sulfurreducens, Fe(III) oxides
on what comprises these structures, the rates of electron seem to preferentially associate with the pili, deleting the pilA
transfer through these structures, and the features that confer gene inhibits Fe(III) oxide reduction, and expressing the PilA
conductivity. gene in trans in the PilA-deficient mutant restores the capacity
Mechanistic investigations linking microbial nanowires to for Fe(III) oxide reduction (Reguera et al., 2005). This is all
mechanisms for extracellular electron transfer have only been consistent with the nanowires being the final conduit, but
carried out with Shewanella oneidensis (Gorby et al., 2006) and does not prove it. In S. oneidensis, there is the observation that
Geobacter sulfurreducens (Reguera et al., 2005). In the case of magnetite accumulates on the surface of some extracellular
S. oneidensis (Gorby et al., 2006), it is not clear what protein(s) material when Fe(III) oxide is reduced, but the nature of this
are the scaffold of the conductive filaments. They are described extracellular material was not determined and there was significant
as ‘pilus-like appendages’ but S. oneidensis may produce a amounts of magnetite associated with other non-filamentous
variety of pili and pseudo-pili, some of which appear to be portions of the cells (Gorby et al., 2006). Another line of
nonconductive (Reguera et al., 2005; Gorby et al., 2006). evidence for electron transfer via nanowires in S. oneidensis is
The conductive structures in S. oneidensis are much thicker that deleting the genes for the cytochromes that appear to make
(50 to > 150 nm diameter) and much less numerous (c. 1 per the S. oneidensis nanowires conductive diminishes electron
cell in figure 1 of Gorby et al., 2006) than most pili and it has transfer to Fe(III) and electrodes (Gorby et al., 2006). How-
been suggested that they are ‘bundles’ of pili, something ever, it has been reported that these same cytochromes are also
which clearly requires further verification. In G. sulfurreducens, associated with the outer membrane of S. oneidensis as well as
the nature of the structure seems more clear because deleting extracellular material that is different from the nanowires (Shi
the gene for PilA, the structural protein for type IV pili, resulted et al., 2007). If so, then the diminished rates of extracellular
in cells without the nanowires (c. 3–5 nm in diameter), which electron transfer in the cytochrome mutants could just as easily
in the wild-type cells are numerous and localized to one lateral be explained as a consequence of cytochromes that are not
side of the cell (Reguera et al., 2005). Reintroducing the gene associated with filaments directly transferring electrons to
for PilA restored the capacity for nanowire production. It seems Fe(III) oxides and electrodes without mediation of the
like similar genetic studies might help identify the structural nanowires. In fact, this is what has recently been proposed
components of the conductive filaments in S. oneidensis. (Shi et al., 2007).
A pressing question for both the S. oneidensis and the G. In a similar manner, there is an abundance of not only c-type
sulfurreducens nanowires is: what features make them conduc- cytochromes, but also other redox-active proteins, such as
tive? For S. oneidensis it has been suggested that cytochromes multicopper proteins, on the outer surface of G. sulfurreducens
might be associated with the pili because deleting genes for (Lovley et al., 2004; Mehta et al., 2005; Mehta et al., 2006;
outer-surface cytochromes resulted in nonconductive filaments Qian et al., 2007), that are required for Fe(III) oxide reduction
(Gorby et al., 2006). At present, there is no evidence that and are not attached to pili. The interaction of these proteins
cytochromes are associated with the pili of G. sulfurreducens. with extracellular electron acceptors must be better elucidated
Ongoing genetic studies in which the PilA protein of G. sul- before it can be definitively stated that the terminal electron
furreducens is being modified with amino acid substitutions transfer step takes place at the pilin surface. Furthermore, it
may provide insights into the mechanisms for conductivity. must be considered that extrusion of pili may be associated
In the future, more involvement by physicists is going to be with the release of other proteins to outer surface. We are now
required in order to make the sophisticated electrochemical also carefully examining whether deleting the PilA gene has
measurements necessary to understand this phenomenon. impacts on the biochemical composition of the outer cell sur-
We have attempted to further evaluate the mechanisms of con- face beyond taking away the capacity for pilin production.

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Journal compilation © 2008 Blackwell Publishing Ltd
 Extracellular electron transfer 227

Shewanella and Geothrix species that produce electron shuttles

DO SOME DISSIMILATORY METAL-REDUCING
(Newman & Kolter, 2000; Nevin & Lovley, 2002a,b; Lies
MICROORGANISMS HAVE ‘IRON LUNGS’?
et al., 2005; Lanthier et al., 2008), Geobacter species must
Another fascinating observation made by Terrry and colleagues directly contact Fe(III) oxides in order to reduce them (Nevin
is that Shewanella putrefaciens CN32 has the novel capacity & Lovley, 2000). This presents a challenge to Geobacter species
to store iron and manganese minerals intracellularly during in subsurface environments in which Fe(III) sources, such as
dissimilatory Fe(III) or Mn(IV) reduction (Glasauer et al., Fe(III) oxides and structural Fe(III) in clays (Shelobolina et al.,
2002, 2004, 2007). Interestingly, the other Shewanella species 2003, 2006), are heterogeneously dispersed. Once a cell depletes
that have been evaluated do not appear to share this ability the Fe(III) oxide in one locale it must find an alternative
(Glasauer et al., 2007). Terry and colleagues have suggested source of the electron acceptor. The current model is that
that the intracellular iron oxide deposits might function ‘as a Geobacter species are motile and hunt for Fe(III) oxides
reservoir of oxidant’ because they are depleted under sustained (Childers et al., 2002). This is consistent with an analysis of
anoxic conditions (Glasauer et al., 2007). This is an intriguing the microbial community during in situ uranium bioremedia-
concept. tion, which indicated that the metabolically active Geobacter
We have yet to identify similar Fe(III) or Mn(IV) oxide species are highly planktonic in the subsurface during active
deposits in Geobacter species, but have discovered what we Fe(III) oxide reduction (Holmes et al., 2007). However, it
believe is an alternative ‘iron lung’ for Geobacter species presents the quandary of how cells can generate ATP required
(Esteve-Núñez et al., 2008). This is the abundance of iron in for motility as well as cell maintenance when they are in a
the periplasm and outer membrane of Geobacter species, which planktonic state searching for a new source of electron acceptor.
is found in the form of c-type cytochromes. The hypothesis is The electron-accepting capacity of the extracytoplasmic cyto-
that these extracytoplasmic cytochromes can act as capacitors chromes provides a potential short-term solution. Respiration
to store electrons in the periplasm and outer membrane when can continue and then once a new source of Fe(III) is located
external electron acceptors are not immediately available. The the electrons stored in the cytochromes can be discharged
reason that this is significant is that energy conservation from from these mini-capacitors.
organic matter oxidation in Geobacter species is expected to In addition to the direct measurements of the electron
result solely from the transport of electrons across the inner storage capacity of the c-type cytochromes in G. sulfurreducens
membrane with associated proton-pumping, which generates there is substantial circumstantial evidence that is consistent
a proton-motive force that drives ATP formation, flagella with this concept. For example, although genetic studies have
rotation, etc. (Mahadevan et al., 2006). Once the electrons indicated that some c-types are essential for growth of G. sul-
are transferred to the periplasm, subsequent electron transfer furreducens on Fe(III), there is remarkably little conservation
steps to extracellular electron acceptors do not further contribute of c-type cytochromes genes across the six Geobacter species
to the cellular energy yield. This is evident from the finding whose genomes have been sequenced. This suggests that there
that Geobacter cell yields are comparable with soluble and has not been evolutionary pressure to maintain specific struc-
insoluble forms of Fe(III), even though there are substantial tures that might promote interaction of the cytochromes with
differences in the redox potentials of these electron acceptors. electron acceptors. However, there has apparently been evo-
The external electron acceptors merely function to oxidize the lutionary pressure for the Geobacter species to maintain an
electron acceptors earlier in the electron transfer chain to abundance of hemes. Not only are there an inordinately
permit continued electron transfer across the inner membrane. high number of c-type cytochrome genes in Geobacter species
The electron-accepting capacity of the c-type cytochromes (Methé et al., 2003) (www.jgi.doe.gov), most of these cyto-
in G. sulfurreducens, determined with two independent methods, chromes have multiple hemes (27 is the maximum yet pre-
was estimated to be c. 1.6 × 10−17 mol electrons per cell dicted) giving them relatively high electron-accepting capacity.
(Esteve-Núñez et al., 2008). This capacitance can, in the The vast majority of the cytochrome genes are expressed
absence of an extracellular electron acceptor, potentially (Ding et al., 2006) and the cells are intensely red due to the
support enough inner membrane electron transfer/proton abundance of cytochromes. Further support for the capacitor
pumping for G. sulfurreducens to satisfy its maintenance energy concept is the finding that the expression of many extracyto-
requirements for 8 min or to swim several hundred cell lengths plasmic c-type cytochrome genes is increased during growth
before all the electron-accepting capacity of the hemes is on either synthetic Fe(III) oxides, sediment Fe(III) oxides or
exhausted (Esteve-Núñez et al., 2008). electrodes versus growth on soluble electron acceptors (Hol-
The ability to temporarily maintain active electron transfer mes et al., 2006) (D.E. Holmes and K.P. Nevin, unpublished
across the inner membrane in the absence of an extracellular data). Such a high abundance of cytochromes is not required
electron acceptor may be key to the survival of Geobacter spe- for Fe(III) reduction, as other Fe(III) reducers, such as
cies in the subsurface and the often-noted ability of Geobacter Pelobacter species (Lovley et al., 1995; Haveman et al., 2006)
species to outcompete a diversity of other dissimilatory Fe(III) and the Fe(III)-reducing hyperthermophiles (Vargas et al., 1998;
reducers in such environments. This is because, in contrast to Kashefi & Lovley, 2003; Lovley et al., 2004), reduce Fe(III)

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228 D. R. LOVLEY

just fine with few, if any c-type cytochromes involved. However, et al., 2008). Shewanella species release flavins, most notably
the energetic investment that Geobacter species make in c-type flavin mononucleotide and riboflavin, that can function as
cytochrome production could be very adaptive in providing an shuttles, enhancing current production in microbial fuel cells
‘iron lung’ that permits electron transfer in the temporary (von Canstein et al., 2008). However, an anonymous reviewer
absence of Fe(III) oxides. recently suggested that another option for a shuttle between
Further investigation into the role of intracellular iron, planktonic cells of S. oneidensis and fuel cell anodes may be
whether as Fe(III) oxides or as heme groups, in serving as a membrane vesicles, similar to those described by Schooling
temporary electron acceptor for dissimilatory Fe(III) reducers and Beveridge ( 2006), if these contain appropriate redox-
is clearly warranted. In addition to Shewanella and Geobacter active components. This is an interesting hypothesis that
species, a diversity of other Fe(III) reducers should be investigated should be further investigated.
as phylogenetically distinct organisms may have evolved different The previous finding that Geobacter species do not appear to
strategies. produce an electron shuttle (Nevin & Lovley, 2000), as well
as electrochemical measurements (Daniel Bond, University of
Minnesota, personal communication), suggests that there is a
HOW ARE ELECTRONS PASSED THROUGH
direct electrical connection between cells of Geobacter and
THE BIOFILMS ON THE ANODES MICROBIAL
anode surfaces. Hunter and Beveridge developed a high-
FUEL CELLS?
pressure freeze-substitution transmission electron microscopic
Superficially, this question may appear to be just another method that permitted high-resolution imaging of Pseudomonas
version of the nanowire questions above. However, electron aeruginosa biofilms (Hunter & Beveridge, 2005b). With this
flow to conductive anode surfaces is functionally different than method, it may be possible to image Geobacter anode biofilms
electron transfer to Fe(III) oxide. Recent research has suggested to elucidate how cells might form a conductive network., e.g.
that, in the most effective microbial fuel cells, electron transfer G. sulfurreducens requires pili to form the thick biofilms on
must proceed through biofilms, a phenomenon for which anodes that yield high power densities in microbial fuel cells
there is little prior guidance in the literature. As detailed below, (Reguera et al., 2006; Nevin et al., 2008b). Do pili from
novel techniques for studying biofilms, such as those recently multiple cells intertwine to promote cell-to-cell electron transfer
developed by Terry and colleagues, could aid in understanding so that cells at substantial distance from the anode surface can
this special form of extracellular electron transfer. establish electrical contact with the anode? Alternatively, pili
Although Fe(III) oxides and the anodes of microbial fuel may play a primarily structural role (Reguera et al., 2007) and
cells both represent insoluble, extracelular electron acceptors, one or more of the multiple redox-active proteins displayed on
Fe(III) oxide sources, and the clays that contain structural Fe(III) the outer surface of G. sulfurreducens may interact to facilitate
that is microbially reducible, are typically smaller than Fe(III)- electron transfer through the biofilm (Nevin et al., 2008b).
reducing microorganisms. Certainly in culture, and possibly in It is important to know how these outer-surface proteins are
sedimentary environments, it may be more correctly stated distributed throughout the biofilm to determine whether they
that the Fe(III) becomes attached to the microbe rather than might contribute to a conductive network. Furthermore, it
vice versa. Furthermore, even when carbon sources are being should be possible to measure the conductivity of the biofilm
added to sedimentary environments, such as during in situ and via genetic manipulation of the quantities of potential
uranium bioremediation, the density of cells is relatively low electron transfer facilitators, identify the key components.
and therefore, organized, confluent biofilms are not expected Such studies are underway.
(Nevin & Lovley, 2002b). In contrast, electrodes represent a The flux of nutrients into, and end products out of, anode
large surface for cell attachment and microbial fuel cells biofilms is likely to significantly impact on the function of
producing high current densities provide high concentrations microbial fuel cells. One obvious concern is delivering fuel
of electron donors. This results in the formation of relatively in an optimum manner. Less obvious, but probably equally
thick (up to c. 75 µm), structured biofilms that are essential for important, is the diffusion of end products out of the biofilm.
high-density current production in Geobacter-powered microbial Protons are of particular concern because extracellular electron
fuel cells (Reguera et al., 2006; Nevin et al., 2008a). S. oneidensis transfer results in substantially more proton release into the
does not build up thick biofilms on anodes, but there is environment that most forms of intracellular reduction of
substantial growth of planktonic cells in S. oneidensis fuel cells electron acceptors (Mahadevan et al., 2006). Accumulation of
(Lanthier et al., 2008). Thus, in both instances most of the these protons may be detrimental for two reasons. The obvious
microorganisms are no longer in close contact with the anode one is that lowering the pH within the biofilm may result in
and in many instances they are far beyond the conceivable suboptimal conditions for continued respiration. Possibly just
reach of nanowires, yet appear to be viable and metabolically as important is the fact that the protons that are produced
active. from substrate oxidation are required on the cathode side of
How is this possible? For S. oneidensis one key component the microbial fuel cell to combine with the electrons released
appears to be soluble electron shuttle molecules (Lanthier from fuel oxidation, providing charge balance. In the near future

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Journal compilation © 2008 Blackwell Publishing Ltd
 Extracellular electron transfer 229

we plan to evaluate the potential for proton accumulation relative rates of metabolism in the subsurface under different
under various fuel cell design strategies with a pH-sensitive conditions imposed during bioremediation. The ultimate
fluoroprobe developed by Hunter and Beveridge (2005a). goal is to be able to predict how dissimilatory metal-reducing
With this probe and confocal scanning laser microscopy, it is microorganisms will behave under different possible bioreme-
possible to determine the pH within the microenvironments diation strategies, making it possible to select an optimized
of biofilms. approach prior to implementing field trials (Lovley, 2003;
Lovley et al., 2008). One of the first steps toward this goal was
the development of a genome-scale in silico model of the
WHAT IS THE PHYSIOLOGICAL STATE OF
metabolism of G. sulfurreducens, which demonstrated that it
METAL-REDUCING MICROORGANISMS
is possible to predictively model the physiology of a Geobacter
UNDER ENVIRONMENTALLY RELEVANT
species under different environmental conditions (Mahadevan
CONDITIONS?
et al., 2006). Subsequent steps include making an in silico
Terry and colleagues demonstrated that the environmental model that reflects the physiology of the Geobacter species that
conditions under which Fe(III)-reducing microorganisms are predominate in a diversity of subsurface environments, known
cultured may significantly influence their physiology, even as subsurface clade 1 (Holmes et al., 2007), and coupling this
impacting which forms of Fe(III) oxides can serve as electron with geochemical and hydrological models. These studies are
acceptors (Glasauer et al., 2003). S. putrefaciens CN32, grown underway.
in minimal medium designed to more accurately reflect These are, of course, only a few of the many potential lines
environmental conditions in the subsurface, could reduce poorly of inquiry about extracellular electron transfer that may be
crystalline Fe(III) oxide, but not the highly crystalline forms promising in the future. Undoubtedly, direct observation and
which CN32 can reduce if grown in rich media (Glasauer measurement of microbes interacting with minerals and each
et al., 2003). The lack of reduction of the crystalline Fe(III) other will play an important role in answering these questions.
forms in more environmentally relevant media is consistent with One can only hope that someone with the talent and insight
data demonstrating that these minerals tend to persist, even in of Terry will come along to fill this critical niche.
organic-rich sedimentary environments (Phillips et al., 1993).
It is an important concept that we must understand the
environmentally relevant physiological status of Fe(III)-reducing REFERENCES
microorganisms in order to make accurate predictions about Anderson RT, Vrionis HA, Ortiz-Bernad I, Resch CT, Peacock A,
process in environments in which Fe(III) reduction is prevalent. Dayvault R, Marutzky S, Metzler DR, Karp K, Lowe M, White DC,
One additional step toward this goal is growing the organisms Long PE, Lovley DR (2003) Stimulating the in situ activity of
Geobacter species to remove uranium from the groundwater of a
under nutrient-limiting conditions in chemostat cultures (Esteve-
uranium-contaminated aquifer. Applied Environmental
Núñez et al., 2005). Another possibility is to grow pure cultures Microbiology 69, 5884–5891.
in sterilized sediments from the site of interest (D.E. Holmes, von Canstein H, Ogawa J, Shimizu S, Lloyd JR (2008) Secretion of
unpublished data). However, the ultimate is to evaluate the flavins by Shewanella species and their role in extracellular electron
physiological status of the natural populations of Fe(III)- transfer. Applied Environmental Microbiology 74, 615–623.
Childers SE, Ciufo S, Lovley DR (2002) Geobacter metallireducens
reducing microorganisms in situ (Lovley, 2003; Lovley et al.,
accesses Fe(III) oxide by chemotaxis. Nature 416, 767–769.
2008). Coppi MV, O’Nei RA, Leang C, Kaufmann F, Methe BA, Nevin KP,
It is now apparent that this is possible, at least for the many Woodard TL, Liu A, Lovley DR (2007) Involvement of Geobacter
subsurface environments in which Fe(III) reduction is an sulfurreducens SfrAB in acetate metabolism rather than
important process and Geobacter species are the predominant intracellular, respiration-linked Fe(III) citrate reduction.
Microbiology 153, 3572–3585.
Fe(III)-reducing organisms. Genome sequences for a number
Ding YR, Hixson K, Giometti CS, Stanley A, Estev-Nunez A,
of Geobacter species are available and from a combination of Khare T, Tollaksen SL, Zhu W, Adkins JN, Lipton MS, Smith RD,
gene expression, proteomic, and genetic studies, it has been Mester T, Lovley DR (2006) The proteome of the dissimilatory
possible to identify genes, whose level of expression is diagnostic metal-reducing microorganism Geobacter sulfurreducens under
of specific physiological conditions and/or the rates of cell various growth conditions. Biochimica et Biophysica Acta 1764,
1198–1206.
metabolism. When coupled with techniques for quantifying
Esteve-Núñez A, Rothermich MM, Sharma M, Lovley DR (2005)
numbers of gene transcripts in the environment this makes Growth of Geobacter sulfurreducens under nutrient-limiting
it feasible to evaluate the in situ physiological status of the conditions in continuous culture. Environmental Microbiology 7,
microorganisms (Holmes et al., 2004, 2005, 2008; O’Neil 641–648.
et al., 2008) (D. E. Holmes et al. unpublished data). With this Esteve-Núñez A, Sosnik J, Visconti P, Lovley DR (2008) Fluorescent
properties of c-type cytochromes reveal their potential role as an
approach it is possible to determine: what nutrients are likely
extra-cytoplasmic electron sink in Geobacter sulfurreducens.
to be limiting the activity of Geobacter species during in situ Environmental Microbiology 10, 497–505.
bioremediation of uranium-contaminated groundwater; whether Glasauer S, Langley S, Beveridge TJ (2002) Intracellular iron minerals
oxygen, toxic metals, etc. are stressing the organisms; and in a dissimilatory iron-reducing bacterium. Science 295, 117–119.

© 2008 The Author

Journal compilation © 2008 Blackwell Publishing Ltd
230 D. R. LOVLEY

Glasauer S, Weidler PG, Langley S, Beveridge TJ (2003) Controls on Lies DP, Hernandez ME, Kappler A, Mielke RE, Gralnick JA,
Fe reduction and mineral formation by a subsurface bacterium. Newman DK (2005) Shewanella oneidensis MR-1 uses overlapping
Geochimica et Cosmochimica Acta 67, 1277–1288. pathways for iron reduction at a distance and by direct contact
Glasauer S, Langley S, Beveridge TJ (2004) Intracellular manganese under conditions relevant for biofilms. Applied Environmental
granules formed by a subsurface bacterium. Environmental Microbiology 71, 4414–4426.
Microbiology 6, 1042–1048. Lloyd JR, Lovley DR, Macaskie LE (2003) Biotechnological
Glasauer S, Langley S, Boyanov M, Lai B, Kemner K, Beveridge TJ application of metal-reducing microorganisms. Advances in
(2007) Mixed-valence cytoplasmic iron granules are linked to Applied Microbiology 53, 85–128.
anaerobic respiration. Applied Environmental Microbiology 73, Lovley DR (1991) Dissimilatory Fe(III) and Mn(IV) reduction.
993–996. Microbiology Review 55, 259–287.
Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Lovley DR (1993) Dissimilatory metal reduction. Annals of Review of
Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kim SK, Microbiology 47, 263–290.
Culley DE, Reed SB, Romine MF, Saffarini DA, Hill EA, Shi L, Lovley DR (1997) Potential for anaerobic bioremediation of BTEX
Elias DA, Kennedy DW, Pinchuk G, Watanabe K, Ishii S, Logan B, in petroleum-contaminated aquifers. Journal of Industrial
Nealson KH, Fredrickson JK (2006) Electrically conductive Microbiology 18, 75–81.
bacterial nanowires produced by Shewanella oneidensis strain MR-1 Lovley DR (2003) Cleaning up with genomics: applying molecular
and other microorganisms. Proceedings of the National Academy of biology to bioremediation. Nature Review of Microbiology 1,
Sciences of the USA 103, 11358–11363. 35–44.
Gralnick JA, Newman DK (2007) Extracellular respiration. Molecular Lovley DR (2006a) Bug juice: harvesting electricity with
Microbiology 65, 1–11. microorganisms. Nature Review of Microbiology 4, 497–508.
Gralnick JA, Vali H, Lies DP, Newman DK (2006) Extracellular Lovley DR (2006b) Microbial fuel cells: novel microbial physiologies
respiration of dimethyl sulfoxide by Shewanella oneidensis strain and engineering approaches. Current Opinions in Biotechnology 17,
MR-1. Proceedings of the National Academy of Sciences of the USA 327–332.
103, 4669–4674. Lovley DR, Baedecker MJ, Lonergan DJ, Cozzarelli IM, Phillips EJP,
Haveman SA, Holmes DE, Ding YR, Ward JE, DiDonato RJ, Siegel DI (1989) Oxidation of aromatic contaminants coupled to
Lovley DR (2006) c-type cytochromes in Pelobacter carbinolicus. microbial iron reduction. Nature 339, 297–299.
Applied Environmental Microbiology 72, 6980–6985. Lovley DR, Phillips EJP, Gorby YA, Landa ER (1991) Microbial
Holmes DE, Chaudhuri SK, Nevin KP, Mehta T, Methe BA, reduction of uranium. Nature 350, 413–416.
Ward JE, Woodward TL, Webster J, Lovley DR (2006) Microarray Lovley DR, Phillips EJP, Lonergan DJ, Widman PK (1995) Fe(III)
and genetic analysis of electron transfer to electrodes in Geobacter and S° reduction by Pelobacter carbinolicus. Applied Environmental
sulfurreducens. Environmental Microbiology 8, 1805–1815. Microbiology 61, 2132–2138.
Holmes DE, Mester T, O’Neil RA, Adams LA, Larrahando MJ, Lovley DR, Coates JD, Blunt-Harris EL, Phillips EJP, Woodward JC
Glaven R, Sharma M, Ward JE, Nevin KP, Lovley DR (2008) (1996) Humic substances as electron acceptors for microbial
Genes for two multicopper proteins required for Fe(III) oxide respiration. Nature 382, 445–448.
reduction in Geobacter sulfurreducens have different expression Lovley DR, Fraga JL, Blunt-Harris EL, Hayes LA, Phillips EJP,
patterns both in the subsurface and on energy-harvesting Coates JD (1998) Humic substances as a mediator for microbially
electrodes. Microbiology (in press). catalyzed metal reduction. Acta Hydrochimica et Hydrobiologica
Holmes DE, Nevin KP, Lovley DR (2004) In situ expression of nifD 26, 152–157.
in Geobacteraceae in subsurface sediments. Applied Environmental Lovley DR, Holmes DE, Nevin KP (2004) Dissimilatory Fe(III)
Microbiology 70, 7251–7259. and Mn(IV) reduction. Advances in Microbial Physiology 49,
Holmes DE, Nevin KP, O’Neil RA, Ward JE, Adams LA, 219–286.
Woodward TL, Lovley DR (2005) Potential for quantifying Lovley DR, Mahadevan R, Nevin KP (2008) Systems biology
expression of Geobacteraceae citrate synthase gene to assess the approach to bioremediation with extracellular electron transfer. In
activity of Geobacteraceae in the subsurface and on current Microbial Degradation: Genomics and Molecular Biology (Diaz, E.,
harvesting-electrodes. Applied Environmental Microbiology 71, ed.), pp. 71–96. Norfolk, UK: Caister Academic Press.
6870–6877. Mahadevan R, Bond DR, Butler JE, Esteve-Nunez A, Coppi MV,
Holmes DE, O’Neil RA, Vrionis HA, N’Guessan LA, Ortiz-Bernad Palsson BO, Schilling. CH, Lovley DR (2006) Characterization of
I, Larrahando MJ, Adams LA, Ward JA, Nicoll JS, Nevin KP, metabolism in the Fe(III)-reducing organism Geobacter
Chavan MA, Johnson JP, Long PE, Lovley DR (2007) Subsurface sulfurreducens by constraint-based modeling. Applied
clade of Geobacteraceae that predominates in a diversity of Environmental Microbiology 72, 1558–1568.
Fe(III)-reducing subsurface environments. ISME Journal 1, Marshall MJ, Beliaev AS, Dohnalkova AC, Kennedy DW, Shi L,
663–677. Wang Z, Boyanov MI, Lai B, Kemner KM, McLean JS, Reed SB,
Hunter RC, Beveridge TJ (2005a) Application of a pH-sensitive Culley DE, Bailey VL, Simonson CJ, Saffarini DA, Romine MF,
fluoroprobe (CSNARF-4) for pH microenvironment analysis in Zachara JM, Fredrickson JK (2006) c-Type cytochrome-dependent
Pseudomonas aeruginosa biofilms. Applied Environmental formation of U (IV) nanoparticles by Shewanella oneidensis.
Microbiology 71, 2501–2510. PLoS Biology 4, e26B. DOI. 10.1371/journal.pbio.0040268.
Hunter RC, Beveridge TJ (2005b) High-resolution visualization of Mehta T, Coppi MV, Childers SE, Lovley DR (2005) Outer
Pseudomonas aeruginosa PAO1 biofilms by freeze-substitution membrane c-type cytochromes required for Fe(III) and Mn(IV)
transmission electron microscopy. Journal of Bacteriology 187, oxide reduction in Geobacter sulfurreducens. Applied
7619–7630. Environmental Microbiology 71, 8634–8641.
Kashefi K, Lovley DR (2003) Extending the upper temperature limit Mehta T, Childers SE, Glaven R, Lovley DR, Mester T (2006)
for life. Science 301, 934. A putative multicopper protein secreted by an atypical type II
Lanthier M, Gregory KB, Lovley DR (2008) Growth with high secretion system involved in the reduction of insoluble electron
planktonic biomass in Shewanella oneidensis fuel cells. FEMS acceptors in Geobacter sulfurreducens. Microbiology 152, 2257–
Microbiology Letters 278, 29–35. 2264.

© 2008 The Author

Journal compilation © 2008 Blackwell Publishing Ltd
 Extracellular electron transfer 231

Methé BA, Nelson KE, Eisen JA, Paulsen IT, Nelson W, Heidelberg OmcB and OmpB of Geobacter sulfurreducens are outer membrane
JF, Wu D, Wu M, Ward N, Beanan MJ, Dodson RJ, Madupu R, surface proteins. FEMS Microbiology Letters 277, 21–27.
Brinkac LM, Daugherty SC, DeBoy RT, Durkin AS, Gwinn M, Reguera G, McCarthy KD, Mehta T, Nicoll J, Tuominen MT,
Kolonay JF, Sullivan SA, Haft DH, Selengut J, Davidsen TM, Lovley DR (2005) Extracellular electron transfer via microbial
Zafar N, White O, Tran B, Romero C, Forberger HA, Weidman J, nanowires. Nature 435, 1098–1101.
Khouri H, Feldblyum TV, Utterback TR, Van Aken SE, Reguera G, Nevin KP, Nicoll JS, Covalla SF, Woodard T, Lovley DR
Lovley DR, Fraser CM (2003) The genome of Geobacter (2006) Biofilm and nanowire production leads to increased current
sulfurreducens: insights into metal reduction in subsurface in Geobacter sulfurreducens fuel cells. Applied and Environmental
environments. Science 302, 1967–1969. Microbiology 72, 7345–7348.
Nevin KP, Kim B-C, Glaven RH, Johnson JP, Woodard TL, Reguera G, Pollina RB, Nicoll JS, Lovley DR (2007) Possible
Methe BA, DiDonato RJ Jr, Covalla SF, Franks AE, Liu A, non-conductive role of Geobacter sulfurreducens pili nanowires in
Lovley DR (2008b) Differences in physiology between biofilm formation. Journal of Bacteriology 189, 2125–2127.
current-producing and fumarate-reducing biofilms of Geobacter Schooling SR, Beveridge TJ (2006) Membrane vesicles: an
sulfurreducens: identification of a novel outer-surface cytochrome overlooked component of the matrices of biofilms. Journal of
essential for electron transfer to anodes at high current densities. Bacteriology 188, 5945–5957.
(submitted). Shelobolina ES, Anderson RT, Vodyanitskii YN, Yuretich R,
Nevin KP, Lovley DR (2000) Lack of production of Lovley DR (2003) Importance of clay size minerals for Fe(III)
electron-shuttling compounds or solubilization of Fe(III) during respiration in a petroleum-contaminated aquifer. Geobiology 2,
reduction of insoluble Fe(III) oxide by Geobacter metallireducens. 67–76.
Applied Environmental Microbiology 66, 2248–2251. Shelobolina ES, Nevin KP, Blakeney-Hayward JD, Nevin KP,
Nevin KP, Lovley DR (2002a) Mechanisms for accessing insoluble Blakeney-Hayward JD, Johnsen CV, Plaia TW, Krader P,
Fe(III) oxide during dissimilatory Fe(III) reduction by Geothrix Woodard T, Holmes DE, VanPraagh CG, Lovley DR (2006)
fermentans. Applied Environmental Microbiology 68, 2294–2299. Geobacter pickeringii, sp. nov., Geobacter argillaceus, sp. nov. and
Nevin KP, Lovley DR (2002b) Mechanisms for Fe(III) oxide Pelosinus fermentans, gen. nov., sp. nov., isolated from subsurface
reduction in sedimentary environments. Geomicrobiology Journal kaolin lenses. International Journal of Systematic and Evolutionary
19, 141–159. Bacteriology 57, 126–135.
Nevin KP, Richter H, Covalla SF, Johnson JP, Woodard TL, Shelobolina ES, Coppi MV, Korenevsky AA, DiDonato LN,
Orloff AL, Jia H, Zhang M, Lovley DR (2008a) Power output of Sullivan SA, Konishi H, Xu H, Leang C, Butler JE, Kim BC, Lovley
Geobacter sulfurreducens comparable to that observed with mixed DR (2007) Importance of the role of c-type cytochromes for U(VI)
communities in microbial fuel cells. (submitted). reduction by Geobacter sulfurreducens. BMC Microbiology 7, 16.
Newman DK, Kolter R (2000) A role for excreted quinones in Shi L, Squier TC, Zachara JM, Fredrickson JK (2007) Respiration of
intracellular electron transfer. Nature 405, 94 –97. metal (hydr)oxides by Shewanella and Geobacter: a key role for
O’Neil RA, Holmes DE, Coppi MV, Adams LA, Larrahando MJ, multihaem c-type cytochromes. Molecular Microbiology 65, 12–20.
Ward JE, Nevin KP, Woodard T, Vironis HA, N’Guessan AL, Stams AJM, de Bok FAM, Plugge CM, van Eekert MHA, Dolfing J,
Lovley DR (2008) Gene transcript analysis of assimilatory iron Schraa G (2006) Exocellular electron transfer in anaerobic
limitation in Geobacteraceae during groundwater bioremediation. microbial communities. Environmental Microbiology 8, 371–382.
Environmental Microbiology (in press). Thamdrup B (2000) Bacterial manganese and iron reduction in
Phillips E, Lovley DR, Roden EE (1993) Composition of aquatic sediments. Advances in Microbial Ecology 16, 41–84.
non-microbially reducible Fe(III) in aquatic sediments. Vargas M, Kashefi K, Blunt-Harris EL, Lovley DR (1998)
Applied Environmental Microbiology 59, 2727–2729. Microbiological evidence for Fe(III) reduction on early Earth.
Qian X, Reguera G, Mester T, Lovley DR (2007) Evidence that Nature 395, 65–67.

© 2008 The Author

Journal compilation © 2008 Blackwell Publishing Ltd