Dissimilatory Metal Reduction: from
Early Life to Bioremediation
Diverse bacteria and archaea use a novel form of
respiration-oxidizing hydrogen or organic compounds with the reduction
of metals
Derek R. Lovley
It is well known that metals, and iron in particular,
are key components of proteins involved in transferring electrons to
terminal acceptors such as oxygen and sulfate. However, it has only
recently been recognized that metals can serve as terminal electron
acceptors to support the anaerobic growth of microorganisms. Here again,
iron is the most important metal, reflecting the considerable abundance
of insoluble Fe(III) oxides in the Earth's crust, but other metals and
metalloids such as manganese, uranium, chromium, technetium, cobalt,
selenium, and arsenic can also serve as electron acceptors. Microbial
reduction of Fe(III) and the oxidized forms of other metals influences
not only the biogeochemical cycles of these metals, but also the fate of
organic matter and nutrients in a variety of environments.
Figure 1
The use of metals as terminal electron acceptors is
called "dissimilatory metal reduction," distinguishing it from
the reduction of metals associated with metal uptake into cells. A
phylogenetically diverse group of bacteria (Fig. 1) and archaea are
known to conserve energy to support growth by oxidizing hydrogen or
organic compounds with the reduction of Fe(III), and novel Fe(III)-reducing
microorganisms are continually being discovered. However, not all
dissimilatory metal reduction is linked to energy conservation. For
example, our studies demonstrate that sulfate-reducing and methanogenic
microorganisms oxidize hydrogen with the reduction of Fe(III). Although
these microorganisms do not appear capable of growing with Fe(III)
serving as the sole terminal electron acceptor, they may preferentially
reduce Fe(III) over sulfate or carbon dioxide.
Dissimilatory metal reduction is relevant to many
issues, ranging from the beginnings of life on Earth to environmental
remediation, but the study of this process is in its infancy. Of all of
the major forms of anaerobic respiration, it has received the least
attention. However, this field seems poised to develop rapidly,
particularly as the Department of Energy and the National Science
Foundation are now supporting numerous research groups that are
investigating both geochemical and biochemical aspects of dissimilatory
metal reduction.
A Model for Early Microbial Respiration
Figure 2
Geological and microbiological evidence suggests that
Fe(III) reduction was a very early form of respiration on Earth.
Geochemists have proposed that high levels of ultraviolet radiation
produced abundant Fe(III) oxides and H2 on the anoxic, prebiotic Earth
(Fig. 2). Other geological sources of hydrogen were also likely.
Although the oxidation of hydrogen coupled with the reduction of Fe(III)
is energetically favorable, catalysts are required for this reaction at
temperatures at which life can exist. Thus, developing a means for
catalyzing this process would have provided a life form with an
excellent strategy for extracting energy from the early Earth
environment. Michael Russell from the University of Glasgow and
colleagues have proposed that the initial step in the evolution of life
was the formation of inorganic iron-sulfur membranes capable of
oxidizing hydrogen coupled to Fe(III) reduction. Then as organic-based
life evolved from this inorganic proto-life, these earliest forms of
life are likely to have gained energy from the same reaction.
Microbiological evidence supports this scenario. The
physiological properties of the last common ancestor(s) of modern
organisms are typically inferred from the physiology of the most deeply
branching extant microorganisms in the 16S rRNA-based tree of life. All
of these "deep branchers" are hyperthermophilic Archaea and
Bacteria. All of the hyperthermophiles that have been tested can
oxidize hydrogen with the reduction of Fe(III), making this process one
of the most highly conserved features of hyperthermophiles. Thus, the
last common ancestor(s) most likely had the ability to oxidize hydrogen
with the reduction of Fe(III), in agreement with geological
considerations.
Modern Biogeochemical Cycles
In addition to hydrogen, modern dissimilatory Fe(III)-reducing
microorganisms can oxidize a variety of organic compounds to carbon
dioxide, with Fe(III) oxides serving as the sole electron acceptor. For
the most part, such microorganisms are not competitive with fermentative
microorganisms for substrates such as sugars and amino acids, but Fe(III)-reducing
microorganisms can oxidize typical products of fermentative
microorganisms, of which acetate is by far the most important in
sedimentary environments. Furthermore, Fe(III) reducers can also oxidize
aromatic compounds and long-chain fatty acids.
Figure 3
Thus, through cooperative activity, fermentative and
Fe(III)-reducing microorganisms can oxidize complex organic matter in
anoxic sedimentary environments to carbon dioxide (Fig. 3). Mn(IV) can
substitute for Fe(III) in this process. Oxidation of organic matter
coupled to the reduction of Fe(III) and Mn(IV) is a major degradative
process in freshwater and marine aquatic sediments, submerged soils (as
in flooded rice paddies and wetlands), and aquifers. It may also be
important in hot (i.e., 80-100șC) environments, since recent studies in
our laboratory show that some hyperthermophiles can also oxidize
acetate, aromatic compounds, and long-chain fatty acids with Fe(III) as
the electron acceptor.
The production of soluble Fe(II) as the result of
microbial Fe(III) oxide reduction in aquifers has important implications
for water quality. When Fe(II)-rich groundwaters are pumped to the
surface and contact oxygen, Fe(II) is reoxidized to insoluble Fe(III)
oxides, which can clog wells, discolor water, and stain just about
everything they contact. Removing this dissolved Fe(II) is expensive.
High Fe(II) levels are a prevalent groundwater problem worldwide.
Fe(III) and Mn(IV) oxides tenaciously bind many trace
metals and phosphorous. Thus, microbial Fe(III) and Mn(IV) reduction
releases trace metals and phosphorous into pore waters of aquatic
sediments and groundwaters. This process influences the nutrient status
of soils, and can affect primary productivity in freshwater environments
because phosphorous levels often determine whether algal blooms develop.
Dissimilatory metal-reducing microorganisms can
influence the biogeochemical cycles of some trace metals by using these
metals as electron acceptors. For example, many dissimilatory
metal-reducing microorganisms can reduce uranium. U(VI), which is
soluble in natural waters, is reduced to U(IV), which precipitates as
the mineral uraninite. Reductive precipitation of uranium in marine
sediments is an important global sink for uranium entering the ocean,
controlling uranium levels in seawater. Reductive precipitation of
uranium in the subsurface is a common source of uranium ores. Several
dissimilatory metal-reducing microorganisms can use gold as an electron
acceptor, reducing soluble oxidized gold, Au(III), to the insoluble
metallic form Au(0). This mechanism may explain how some gold deposits
have been formed.
Under appropriate conditions, one of the end products of
microbial reduction of Fe(III) oxide is the magnetic mineral magnetite.
Large accumulations of magnetite in Precambrian iron deposits as well as
in the deep, hot subsurface and around hydrocarbon deposits could be
geological signatures of the activity of Fe(III)-reducing
microorganisms.
Bioremediation of Organic and Metal Contaminants
Much recent research on dissimilatory metal-reducing
microorganisms has focused on their role in bioremediating contaminated
subsurface environments. For example, when subsurface environments are
contaminated with organic compounds, such as petroleum or landfill
leachate, anoxic conditions typically develop as microorganisms consume
the small amount of oxygen typically available in groundwater. With the
development of anoxic conditions, Fe(III) is generally the most abundant
potential electron acceptor for organic matter oxidation in the
subsurface. According to geochemical studies, significant amounts of
organic contaminants, such as aromatic hydrocarbons, can be oxidized
within subsurface environments with Fe(III) serving as the electron
acceptor.
Pure cultures of dissimilatory Fe(III)-reducing
microorganisms, such as Geobacter metallireducens, can oxidize a
variety of aromatic contaminants. In fact, G. metallireducens was
the first organism of any kind found to anaerobically oxidize an
aromatic hydrocarbon. Studies with contaminated sediments and enrichment
cultures have demonstrated that Fe(III)-reducing microorganisms can even
oxidize unsubstituted aromatic hydrocarbons, such as benzene and
naphthalene, that were thought to be resistant to anaerobic degradation.
Although Fe(III)-reducing microorganisms can degrade
organic contaminants within polluted aquifers, this process can be slow.
One of the key limiting factors is the rate at which Fe(III)-reducing
microorganisms can access the insoluble Fe(III) oxides in the
subsurface. One strategy for stimulating the activity of Fe(III)
reducers in aquifer sediments is to add humic acids or other quinone-containing
compounds, to which Fe(III)-reducing microorganisms can transfer
electrons. The resulting hydroquinones react spontaneously with Fe(III)
oxides, reducing Fe(III) to Fe(II) and regenerating the quinones to
undergo additional cycles of reduction and oxidation. This electron
shuttling via extracellular quinones can greatly accelerate the rate and
extent of the anaerobic microbial bioremediation of benzene and
additional otherwise refractory contaminants such as methyl-tert-butyl
ether (MTBE), vinyl chloride, and dichloroethene.
Besides controlling the spread of organic contaminants
in the subsurface, dissimilatory metal-reducing microorganisms may also
prevent the migration of metal contaminants in groundwater. Consider the
radioactive metal uranium, which is a major contaminant in many areas
where it has been mined and processed. In many instances, the
contaminated groundwater contains dissolved oxygen and uranium in its
soluble form, U(VI). When a simple organic compound such as acetate is
added to the subsurface, microorganisms metabolize it, quickly consuming
available dissolved oxygen and nitrate. Then dissimilatory
metal-reducing microorganisms begin to metabolize the remaining acetate,
oxidizing it to carbon dioxide while reducing available metals.
Figure 4
Even in uranium-contaminated subsurface environments,
Fe(III) is generally the most abundant metal electron acceptor. However,
while reducing Fe(III), the microbial Fe(III) reducers can also convert
U(VI) to U(IV), which precipitates from the groundwater and is
immobilized in the subsurface (Fig. 4). Laboratory studies and
preliminary field experiments suggest that this mechanism may be
effective for stopping further spread of subsurface uranium
contamination and for concentrating uranium in a discrete zone for
eventual retrieval. Similar approaches are being considered for
immobilizing other radioactive metal contaminants such as technetium,
cobalt, and highly toxic chromium, as well as the metalloid selenium.
Microorganisms Involved in Dissimilatory Metal
Reduction
Many different Fe(III)-reducing microorganisms have been
recovered from a diverse range of aquatic sediments, submerged soils,
and other subsurface environments. The most intensively studied Fe(III)-reducing
microorganisms are Shewanella species, which are in the gamma
subclass of the Proteobacteria. One feature that makes this genus
attractive for study is that cells can be grown to high densities using
oxygen as an electron acceptor and then placed under anoxic conditions
to study reduction of Fe(III).
The complete genome of Shewanella oneidensis
(formerly S. putrefaciens) is now available (www.tigr.org), and Shewanella
species are amenable to genetic studies. However, investigators who have
used unbiased molecular techniques to analyze microbial communities have
consistently found that Shewanella are not significant components
of communities in a wide variety of environments in which Fe(III)
reduction is important. One reason for their relative scarcity is that
their preferred organic electron donors, such as lactate, are not
important intermediates for anaerobic metabolism in sedimentary
environments. Another reason is that, as detailed below, Shewanella
species appear to transfer electrons to Fe(III) oxides in ways that are
not well suited for low-energy, nutrient-poor sedimentary environments.
Microorganisms in the family Geobacteraceae represent
another group of well-studied Fe(III)-reducing microorganisms. This
family is within the delta subclass of the Proteobacteria and
includes the genera Geobacter, Desulfuromonas, Desulfuromusa,
and Pelobacter. Although these organisms were previously
classified as strict anaerobes, recent evidence suggests that they
readily tolerate oxygen exposure. Oxidation of organic matter coupled to
Fe(III) reduction has primarily been studied in Geobacter
species. For instance, Geobacter metallireducens (formerly strain
GS-15) was the first microorganism found to completely oxidize organic
compounds to carbon dioxide with Fe(III) or other metals serving as the
electron acceptor.
One of the most environmentally relevant organic
electron donors that G. metallireducens and other Geobacter species
oxidize is acetate, a key intermediate in the anaerobic metabolism of
organic matter in sedimentary environments. Furthermore, G.
metallireducens provides a pure culture model for the oxidation of
aromatic contaminants coupled to the reduction of Fe(III) in subsurface
environments, as it can also oxidize a variety of aromatic compounds,
including important contaminants such as phenol, p-cresol, and even the
aromatic hydrocarbon toluene, with Fe(III) as the electron acceptor.
Interest in Geobacteraceae has increased as
molecular analyses have indicated that Geobacteraceae are
significantly enriched in a variety of sedimentary environments in which
dissimilatory metal reduction is an important process. This was first
observed in a petroleum-contaminated aquifer in which Fe(III)-reducing
microorganisms were removing benzene and other aromatic hydrocarbon
contaminants from the groundwater. Similar enrichments of Geobacteraceae
were observed in other aquifers in which the introduction of petroleum
or other organic compounds led to development of Fe(III)-reducing
conditions. For instance, Geobacteraceae accounted for more than
40% of the microbial community when organic compounds were added to
promote U(VI) reduction in uranium-contaminated aquifer sediments.
Studies by Kenneth Nealson and colleagues at California Institute of
Technology on aquatic sediments, and by H.W. van Vereveld at the
University of Amsterdam on groundwater contaminated with landfill
leachate, have similarly found a predominance of Geobacteraceae
under conditions in which Fe(III) reduction is important.
The availability of pure cultures closely related to the
Geobacteraceae that live in subsurface environments provides a
rare opportunity in environmental microbiology to study an
environmentally relevant organism under defined laboratory conditions.
Thus, the Geobacteraceae provide an excellent opportunity not
only to learn more about the factors controlling the rate and extent of
microbial metal reduction in the environment, but also to elucidate
other physiological properties that might help microorganisms colonize
anoxic environments.
The genomic sequence of one member of the Geobacteraceae,
G. sulfurreducens, is available (www.tigr.org), and researchers
at the Joint Genome Institute (www.jgi.doe.gov) have completed draft
genomic sequences of G. metallireducens and Desulfuromonas
acetoxidans. A genetic system for G. sulfurreducens has been
developed, and whole-genome DNA microarrays are being constructed.
Coupled with proteomics investigations and more traditional biochemical
studies, these approaches are expected to lead to a much better
understanding of Geobacter physiology in the near future.
A diverse collection of other mesophilic Fe(III)-reducing
microorganisms has been recovered from a variety of environments, but
less information is available on their distribution and physiology. For
example, in some instances molecular analysis has detected
microorganisms closely related to Geothrix fermentans in the
Fe(III)-reducing zone of aquifer sediments, but always orders of
magnitude less than the Geobacteraceae.
Of the thermophilic and hyperthermophilic Fe(III)
reducers, the archaea Geoglobus ahangari and Ferroglobus
placidus are of special interest. These are the first
hyperthermophiles documented to anaerobically oxidize acetate with any
electron acceptor. With Fe(III) as the electron acceptor, F. placidus
can also oxidize aromatic compounds, and G. ahangari can oxidize
long-chain fatty acids. The discovery of these forms of metabolism in
hyperthermophiles greatly expands the known metabolic diversity of Archaea
and provides evidence that organic matter may be anaerobically oxidized
in hot ecosystems in a manner similar to that previously described for
more temperate environments.
Mechanisms for Metal Reduction
The biochemical mechanisms for Fe(III) reduction are
beginning to be understood. Thomas DiChristina and collaborators at the
Georgia Institute for Technology showed that the export of key electron
transport components, such as c-type cytochromes, to the outer membrane
is essential for Fe(III) reduction in S. oneidensis. Other
studies, most notably by Charles Myers and collaborators at the Medical
College of Wisconsin in Milwaukee, Wis., demonstrated that c-type
cytochromes are essential intermediates in electron transfer to metals
in this organism. However, the terminal metal reductases are not yet
identified. Genetic studies in our laboratory demonstrated that c-type
cytochromes are involved in Fe(III) and U(VI) reduction in G.
sulfurreducens, and earlier biochemical studies suggested that the
c3 cytochrome is the U(VI) reductase in Desulfovibrio vulgaris.
Figure 5
Some Fe(III)-reducing microorganisms reduce Fe(III)
without directly contacting the Fe(III) oxide--a finding that overturns
a longstanding assumption that direct contact is necessary (Fig. 5).
After determining that S. oneidensis releases quinones into the culture
medium during growth, Dianne Newman, now at California Institute for
Technology in Pasadena, Calif., and Roberto Kolter of Harvard University
in Cambridge, Mass., suggested that quinones might serve as an electron
shuttle between S. oneidensis and Fe(III) oxide.
Meanwhile, by sequestering Fe(III) oxides within
microporous alginate beads, which only admit molecules that are smaller
than 12 kDa, my colleagues and I confirmed that Shewanella alga—and
also Geothrix fermentans—reduce Fe(III) oxides without directly
contacting them. Although our preliminary evidence suggests that
quinones serve as the electron shuttles, the precise nature of the
shuttle molecules remains to be determined. In addition to producing
electron shuttling compounds, both S. alga and G. fermentans solubilize
Fe(III) during growth on insoluble Fe(III) oxide, presumably by
releasing one or more Fe(III)-chelating compounds--representing another
mechanism for Fe(III)-reducing microorganisms to overcome the need for
direct contact with Fe(III) oxides.
In contrast, Geobacter metallireducens does not
release electron shuttling compounds or solubilize Fe(III) from Fe(III)
oxide and must contact Fe(III) oxide to reduce it. G. metallireducens
is highly adapted for this mode of Fe(III) oxide reduction. When growing
on soluble electron acceptors, including chelated Fe(III), it is
nonmotile. However, when only insoluble Fe(III) or Mn(IV) oxides are
available as electron acceptors, G. metallireducens produces
flagella and is chemotactic to Fe(II) and Mn(II), following the gradient
of these metals that emanate from Fe(III) and Mn(IV) oxides under anoxic
conditions. Pili are also specifically produced during growth on the
oxides and are necessary for Fe(III) oxide reduction, presumably serving
as a means of attachment to the oxides. G. metallireducens'
strategy for the reduction of Fe(III) and Mn(IV) oxides is considered to
be a more energetically effective mechanism than the production of
electron shuttles and Fe(III) chelators under the conditions typically
found in subsurface environments, which may explain the predominance of Geobacteraceae
in such environments.
Harvesting Electricity with Metal Reducers
The fact that we are still only beginning to realize the
unique capabilities of dissimilatory metal-reducing microorganisms is
clear from our recent finding that Geobacteraceae are useful for
harvesting energy from the environment in the form of electricity. Lenny
Tender of the Naval Research Laboratory, Washington, D.C., and Clare
Reimers of Oregon State University in Corvallis found that placing a
graphite electrode in anaerobic marine sediments and connecting it to
similar electrode in the overlying water yielded electrical current. We
found, in studies supported by the Office of Naval Research, that the
surface of the electrodes in the sediments were highly enriched in Geobacteraceae
and that members of this family could produce electricity and grow
in pure culture with organic compounds as the electron donor and a
graphite electrode as the sole electron acceptor. Although the immediate
application of this technology is for the deployment of electronic
monitoring devices in the ocean, with further optimization Geobacteraceae
living on electrodes have significant potential for harvesting
energy from a variety of organic wastes and in bioremediation.
SUGGESTED READING
Bond, D. R., D. E. Holmes, L. M. Tender,
and D. R. Lovley. 2002. Electrode-reducing
microorganisms harvesting energy from marine sediments. Science 295:483-485.
Childers, S. E., S. Ciufo, and D. R.
Lovley. Geobacter metallireducens access
Fe(III) oxide by chemotaxis. Nature, in press.
DiChristina, T. J., C. M. Moore, and C.
A. Haller. 2002. Dissimilatory Fe(III) and
Mn(IV) reduction by Shewanella putrefaciens requires ferE,
a homolog of the pulE (gspE) type II protein secretion
gene. J.
Bacteriol. 184:142-151.
Lovley, D. R. 2000.
Fe(III)- and Mn(IV)-reducing prokaryotes, www.prokaryotes.com. In M.
Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt
(ed.), The prokaryotes. Springer-Verlag, Inc., New York.
Lovley, D. R. 2000. Fe(III)
and Mn(IV) reduction, p. 3-30. In D. R. Lovley (ed.),
Environmental microbe-metal interactions. ASM Press, Washington, D.C.
Myers, J. M., and C. R. Myers. 2000.
Role of the tetraheme cytochrome CymA in anaerobic electron transport in
cells of Shewanella putrefaciens MR-1 with normal levels of
menaquinone. J.
Bacteriol. 182:67-75.
Newman, D. K., and R. Kolter. 2000.
A role for excreted quinones in extracellular electron transfer. Nature 405:94-97.
Search | Site Map | Home
