Dissimilatory Fe(III) and Mn(IV) reducing prokaryotes

Dissimilatory Fe(III)- and Mn(IV)-Reducing
Prokaryotes
DEREK LOVLEY
Introduction
Dissimilatory Fe(III) reduction is the process in which microorganisms transfer
electrons to external ferric iron [Fe(III)], reducing it to ferrous iron [Fe(II)] without
assimilating the iron. A wide phylogenetic diversity of microorganisms, including
archaea as well as bacteria, are capable of dissimilatory Fe(III) reduction. Most
microorganisms that reduce Fe(III) also can transfer electrons to Mn(IV), reducing it
to Mn(II).
As detailed in the next section, dissimilatory Fe(III) and Mn(IV) reduction is one of
the most geochemically significant events that naturally takes place in soils, aquatic
sediments, and subsurface environments. Dissimilatory Fe(III) and Mn(IV) reduction
has a major influence not only on the distribution of iron and manganese, but also on
the fate of a variety of other trace metals and nutrients, and it plays an important
role in degradation of organic matter. Furthermore, dissimilatory Fe(III)-reducing
microorganisms show promise as useful agents for the bioremediation of
sedimentary environments contaminated with organic and/or metal pollutants.
Despite their obvious environmental significance, Fe(III) and Mn(IV)-reducing
microorganisms are among the least studied of any of the microorganisms that carry
out important redox reactions in the environment.
The Fe(III)- and Mn(IV)-reducing microorganisms are also of intrinsically interesting
because they have unique metabolic characteristics. Foremost is the ability of these
microorganisms to transfer electrons to external, highly insoluble electron acceptors
such as Fe(III) and Mn(IV) oxides, as well as extracellular organic compounds such
as humic substances. Furthermore, microbiological and geological evidence suggests
that dissimilatory Fe(III) reduction was one of the earliest forms of microbial
respiration. Thus, insights into Fe(III) reduction mechanisms may aid in
understanding the evolution of respiration in microorganisms.
Significance of Fe(III)- and Mn(IV)-
reducing Microorganisms

Some claims for the significance of Fe(III)-reducing microorganisms may be
exaggerated, such as the assertion that "if it were not for the bacterium GS-15 [a
Fe(III)-reducing microorganism] we would not have radio and television today"
(Verschuur, 1993). However, it is also clear that Fe(III)-reducing microorganisms are
of vitally important to the proper functioning of a variety of natural ecosystems and
have practical applications. Detailed reviews of the literature covering many of these
aspects of Fe(III) and Mn(IV) reduction are available (Lovley, 1987a; Lovley, 1991a;
Lovley, 1993a; Nealson and Saffarini, 1994; Lovley, 1995a; Lovley et al. 1997c).
Therefore only highlights of the significance of Fe(III)-reducing microorganisms,
abstracted from these reviews, will be briefly summarized here.
Oxidation of Organic Matter in Anaerobic
Environments
Microbial oxidation of organic matter coupled to the reduction of Fe(III) and Mn(IV)
is an important mechanism for organic matter oxidation in a variety of aquatic
sediments, submerged soils, and in aquifers. Depending on the aquatic sediments or
submerged soils considered, Fe(III) and/or Mn(IV) reduction have been estimated to
oxidize anywhere from 10% to essentially all of the organic matter oxidation in the
sediments (Lovley, 1991a; Canfield et al., 1993; Lovley, 1995b; Lovley et al.,
1997c). An important factor that enhances the significance of Fe(III) and Mn(IV)
reduction in aquatic sediments is bioturbation which leads to the reoxidation of Fe(II)
and Mn(II) so that each molecule of iron and manganese can be used as an electron
acceptor multiple times prior to permanent burial. In deep pristine aquifers, there
are often extensive zones exist in which Fe(III) reduction is the predominant
mechanism for organic matter oxidation (Chapelle and Lovley, 1992; Lovley and
Chapelle, 1995c). The ability of Fe(III)-reducing microorganisms to outcompete
sulfate-reducing and methanogenic microorganisms for electron donors during
organic matter degradation is an important factor limiting the production of sulfides
and methane in some submerged soils, aquatic sediments, and the subsurface
(Lovley, 1991a; Lovley, 1995b).
A model for the oxidation of organic matter in sedimentary environments in which

Fe(III) reduction is the predominant terminal electron-accepting process has been
suggested (Lovley et al., 1997c). This model is based upon the known physiological
characteristics of Fe(III)- and Mn(IV)-reducing microorganisms available in pure
culture as well as on studies on the metabolism of organic matter metabolism by
natural communities of microorganisms living in various sedimentary environments
in which Fe(III) reduction is the terminal electron-accepting process (TEAP). In this
model (Fig. 1), complex organic matter is hydrolyzed to simpler components by the
action of hydrolytic enzymes from a variety of microorganisms. Fermentative
microorganisms are the principal consumers of fermentable compounds such as
sugars and amino acids and these compounds are converted primarily to
fermentation acids and, possibly to hydrogen. Acetate is by far the most important
fermentation acid produced (Lovley and Phillips, 1989a). Acetate also may be
produced as the result of incomplete oxidation of some sugars by some Fe(III)-
reducing microorganisms (Coates et al., 1999a). Other Fe(III)-reducing
microorganisms oxidize the acetate and other intermediary products. Some Fe(III)-
reducing microorganisms also can oxidize aromatic compounds and long-chain fatty
acids. Thus, through the activity of diverse microorganisms, complex organic matter
can be oxidized to carbon dioxide with Fe(III) serving as the sole electron acceptor.
A similar model probably is probably appropriate for organic matter oxidation in
sediments in which Mn(IV) reduction is the TEAP. This model emphasizes that
acetate is likely to be the major electron donor for Fe(III) or Mn(IV) reduction in
environments in which naturally occurring, complex organic matter is the major
substrate for microbial metabolism. However, when otherwise organic-poor
environments, such as sandy aquifers, are contaminated with a specific class of
organic compounds, such as aromatics, then these contaminants may be the most
important direct electron donors for Fe(III) or Mn(IV) reduction.
Fig. 1. Proposed pathways for organic matter degradation in mesophilic
environments in which Fe(III) reduction is the predominant terminal electron-
accepting process.
Influence on Metal and Nutrient Geochemistry and Water Quality

The reduction of Fe(III) to Fe(II) is one of the most important geochemical changes
as anaerobic conditions develop in submerged soils and aquatic sediments
(Ponnamperuma, 1972). The Fe(II) produced as the result of Fe(III) reduction is the
primary reduced species responsible for the negative redox potential in many
anaerobic freshwater environments. The reduction of Fe(III) oxides and of the
structural Fe(III) in clays typically results in a change in soil color from the red-
yellow of Fe(III) forms to the green-gray of Fe(II) minerals (Lovley, 1995c). The
oxides of Fe(III) and Mn(IV) oxides bind trace metals, phosphate, and sulfate, and
Fe(III) and Mn(IV) reduction is associated with the release of these compounds into
solution (Lovley, 1995a). Also, typically the pH, ionic strength of the pore water, and
the concentration of a variety of cations are increased (Ponnamperuma, 1972;
1984). All of these changes influence water quality in aquifers and can affect the
growth of plants in soils.
The solubility of Fe(II) and Mn(II) is greater than that of Fe(III) and Mn(IV) and thus
Fe(III) and Mn(IV) reduction result in an increase in dissolved iron and manganese in
pore waters. Undesirably high concentrations of iron and manganese may be toxic to
plants (Lovley, 1995b) and are particularly significant in groundwaters sources of
drinking water, being one of the most prevalent groundwater quality problems
(Anderson and Lovley, 1997).
Most of the Fe(II) and Mn(II) produced from microbial Fe(III) and Mn(IV) reduction
is found in solid phases, often in the form of Fe(II) and Mn(II) minerals of
geochemical significance (Lovley, 1995c). The most intensively studied mineral that
is formed during microbial Fe(III) reduction is the magnetic mineral magnetite
(Fe
3
O
4
) (Lovley et al., 1987c; Lovley, 1990a; Lovley, 1991a). The magnetite
produced during microbial Fe(III) reduction can be an important geological signature
of this activity. For example, large quantities of magnetite at depths up to 6.7 km

below the Earth's surface provided some of the first evidence for a deep, hot
biosphere (Gold, 1992). The massive magnetite accumulations that comprise the
Precambrian Banded Iron Formations provide evidence for the possible activity of
Fe(III)-reducing microorganisms on early Earth. Formation of magnetite as the result
of microbial Fe(III) reduction may contribute to the magnetic remanence of soils and
sediments. The magnetic anomalies that aid in the localization of subsurface
hydrocarbon deposits may result from the activity of hydrocarbon-degrading Fe(III)
reducers. Formation of other Fe(II) and Mn(II) minerals such as siderite (FeCO
3
) and
rhodochrosite (MnCO
3
) also may provide geological signatures of microbial Fe(III)
and Mn(IV) reduction.
As detailed below, many Fe(III)- and Mn(IV)-reducing microorganisms can use other
metals and metalloids as electron acceptors. Microbial reduction of the soluble
oxidized form of uranium, U(VI), to insoluble U(IV) may be an important mechanism
for the formation of uranium deposits and the reductive sequestration of uranium in
marine sediments, the process which prevents dissolved uranium from building up in
marine waters (Lovley et al., 1991a; Lovley and Philips 1992). Reduction of other
metals such as vanadium, molybdenum, copper, gold, and silver, as well as
metalloids such as selenium and arsenic, can affect the solubility and fate of these
compounds in a variety of sedimentary environments and may contribute to ore
formations (Lovley, 1993a; Oremland, 1994a; Newman et al., 1998; Kashefi and
Lovley, 1999).
Bioremediation of Organic and Metal Contaminants
Iron [Fe(III)]-reducing microorganisms have been shown to play a major role in
removing organic contaminants from polluted aquifers. For example, Fe(III)-reducing
microorganisms naturally remove aromatic hydrocarbons from petroleum-
contaminated aquifers (Lovley et al., 1989b; Lovley, 1995c; Lovley, 1997a;

Anderson et al., 1998) and this process can be artificially enhanced with compounds
that make Fe(III) more available for microbial reduction (Lovley et al., 1994a;
Lovley, 1997a). The Fe(II)-minerals formed as the result of microbial Fe(III)
reduction can be important reductants for the reduction of nitroaromatic
contaminants (Heijman et al., 1993; Hofstetter et al., 1999). Minerals containing
Fe(II) also may serve to reductively dechlorinate some chlorinated contaminants
(Fredrickson and Gorby, 1996).
The ability of Fe(III)-reducing microorganisms to substitute other metals and
metalloids in their respiration may be exploited for remediation of metal
contamination (Lovley, 1995a; Lovley, 1995b; Fredrickson and Gorby, 1996; Lovley
and Coates, 1997b). Reduction of soluble U(VI) to insoluble U(IV) can effectively
precipitate uranium from contaminated groundwaters and surface waters. Microbial
uranium reduction can be coupled with a simple soil-washing procedure to
concentrate uranium from contaminated soils. Iron [Fe(III)]-reducing
microorganisms can precipitate technetium from contaminated waters by reducing
soluble Tc(VII) to insoluble Tc(IV). Soluble radioactive Co(III) complexed to EDTA
can be reduced to Co(II) which is less likely to be associated with the EDTA found in
contaminated groundwaters and more likely to adsorb to aquifer solids. Some Fe(III)
reducers convert soluble, toxic Cr(VI) to less soluble less toxic Cr(III). Reduction of
soluble selenate to elemental selenium can effectively precipitate selenium in
sediments or remove selenate from contaminated waters in bioreactors.
A Possible Early Form of Microbial Respiration
Iron [Fe(III)] reduction may have been one of the earliest forms of microbial
respiration (Vargas et al., 1998). Biological evidence for this hypothesis is the finding
from 16S rRNA phylogenies that all of microorganisms that are the most closely
related to the last common ancestor of extant microorganisms are Fe(III)-reducing
microorganisms. All of the deeply branching bacteria and archaea that have been
examined can oxidize hydrogen with the reduction of Fe(III). Several that have been
examined in more detail can conserve energy to support growth from this
metabolism. Of most interest in this regard is Thermotoga maritima, which was

previously considered to be a fermentative organism because it could not conserve
energy to support growth from the reduction of other commonly considered electron
acceptors. However, T. maritima it does grow via Fe(III) respiration. This result and
the apparent conservation of the ability to reduce Fe(III) in all these deeply
branching organisms suggests that the last common ancestor was a hydrogen-
oxidizing, Fe(III)-reducing microorganism.
The concept that Fe(III) reduction is an early form of respiration agrees with
geological scenarios that suggest the presence of large quantities of Fe(III) on
prebiotic Earth (Cairns-Smith et al., 1992; de Duve, 1995) and elevated hydrogen
levels (Walker, 1980)—conditions that would be conducive to the evolution of a
hydrogen-oxidizing, Fe(III)-reducing microorganism. The large accumulations of
magnetite in the Precambrian iron formations (discussed above) indicate that the
accumulation of Fe(III) on prebiotic Earth was biologically reduced early in the
evolution of life on Earth. This and other geochemical considerations suggest that
Fe(III) reduction was the first globally significant mechanism for organic matter
oxidation (Walker, 1987; Lovley, 1991a).
Fe(III)- and Mn(IV)- reducing Microorganisms
Available in Pure Culture
Dissimilatory Fe(III)- and Mn(IV)-reducing microorganisms can be separated into
two major groups, those that support growth by conserving energy from electron
transfer to Fe(III) and Mn(IV) and those that do not. Early investigations on Fe(III)
and Mn(IV) reduction in pure culture were conducted exclusively with organisms that
are not considered to be conservers of energy from Fe(III) or Mn(IV) reduction
(Lovley, 1987a). However, within the last decade, a diversity of microorganisms has
been described in which Fe(III) and Mn(IV) reduction are linked to respiratory
systems capable of ATP generation. It is these Fe(III)- and Mn(IV)-respiring
microorganisms (abbreviated here as FMR) that are likely to be responsible for most
of the Fe(III) and Mn(IV) reduction in many sedimentary environments (Lovley,
1991a). A brief description of the known metabolic and phylogenetic diversity of
dissimilatory Fe(III)- and Mn(IV)-reducing microorganisms follows.

Fermentative Fe(III)- and Mn(IV)- reducing Microorganisms
Many microorganisms which grow via fermentative metabolism can use Fe(III) or
Mn(IV) as a minor electron acceptor during fermentation (Table 1). Growth is
possible in the absence of Fe(III) or Mn(IV). In this form of Fe(III) and Mn(IV)
reduction, most of the electron equivalents in the fermentable substrates are
recovered in organic fermentation products and hydrogen. Typically, less than 5% of
the reducing equivalents are transferred to Fe(III) or Mn(IV) (Lovley, 1987a; Lovley
and Phillips, 1988b). However, significant amounts of Fe(II) and Mn(II) can
accumulate in cultures of these fermentative organisms when Fe(III) or Mn(IV) is
provided as a potential electron sink. Although thermodynamic calculations have
demonstrated that fermentation with Fe(III) reduction [electron transfer to Fe(III)]
is more energetically favorable than fermentation without Fe(III) reduction (Lovley
and Phillips, 1989a), it has not been demonstrated that the minor transfer of
electron equivalents to Fe(III) or Mn(IV) during fermentation causes any increase in
cell yield. In contrast to these fermentative microorganisms, several microorganisms
can partially or completely oxidize fermentable sugars and amino acids with the
reduction of Fe(III) and conserve energy from this metabolism, as discussed below.
Table 1. Organisms known to reduce Fe(III) but not known to conserve energy from
Fe (III) reduction.
Sulfate- reducing Microorganisms
Many respiratory microorganisms that grow anaerobically with sulfate serving as the
electron acceptor also have the ability to enzymatically reduce iron [Fe(III); Table
1]. Electron donors that support Fe(III) reduction are the same ones that support
sulfate reduction by sulfate-reducing microorganisms. However, none of these
sulfate reducers have been shown to grow with Fe(III) serving as the sole electron
acceptor (Lovley et al., 1993b). This is true despite the fact that sulfate reducers
have a higher affinity for hydrogen, and possibly for other electron donors, than for
sulfate when Fe(III) serves as the electron acceptor (Coleman et al., 1993; Lovley et
al., 1993c).

The advantage to sulfate reducers in reducing Fe(III), if there is one, has not been
thoroughly investigated. Because it has been found that the intermediate electron
carrier, cytochrome c
3
, can function as an Fe(III) reductase (Lovley et al., 1993),
intermediate electron carriers involved in sulfate reduction may inadvertently reduce
Fe(III) because it has been found that the intermediate electron carrier, cytochrome
c
3
can function as an Fe(III) reductase (Lovley et al., 1993b). Alternatively, Fe(III)
reduction by sulfate reducers may be a strategy to hasten Fe(III) depletion and
enhance conditions for sulfate reduction. Furthermore, the possibility that sulfate-
reducing microorganisms may be able to generate ATP as the result of Fe(III)
reduction, even if they can not grow with Fe(III) as the sole electron acceptor, has
not been ruled out (Lovley et al., 1993c).
In contrast to the sulfate-reducing microorganisms discussed above, which could not
be grown with Fe(III) as the sole electron acceptor, it has been suggested (Tebo and
Obraztsova, 1998) that the sulfate-reducing microorganism "Desulfotomaculum
reducens" could also conserve energy to support growth by reducing Fe(III), Mn(IV),
U(VI), and Cr(VI) (Tebo and Obraztsova, 1998). However, the data supporting the
claim that energy is gained from electron transport to metals is curious. For
example, when the culture was grown on 400 μM U(VI), the cell yield was greater
than when the culture reduced 8 mmol Fe(III). This occurs despite the fact that the
number of electrons transferred to Fe(III) was ten-fold higher than the electron
transfer to U(VI) and that Fe(III) reduction is energetically more favorable than
U(VI) reduction. Cell yields with metals as the electron acceptor were comparable to
those during sulfate reduction even though electron transfer to sulfate was at least
250-fold, and in some instances 2500-fold, greater than electron transfer to the
metals. These results suggest that the presence of the metals had some additional
influence on growth other than just serving as an electron acceptor.

Several sulfate-reducing microorganisms can oxidize S° to sulfate, with Mn(IV)
serving as the electron acceptor, but were not found to conserve energy to support
growth from this reaction (Lovley and Phillips, 1994a). Enrichment cultures that are
established at circumneutral pH with S° as the electron donor and Mn(IV) or Fe(III)
as the electron acceptor typically yield microorganisms which that disproportionate
S° to sulfate and sulfide (Thamdrup et al., 1993). The Fe(III) or Mn(IV) serve to
abiotically reoxidize the sulfide produced.
Microorganisms that Conserve Energy to Support
Growth from Fe(III) and Mn(IV) Reduction
The Fe(III)- and Mn(IV)-respiring microorganisms (FMR) which are known to
conserve energy to support growth from Fe(III) and Mn(IV) reduction (Table 2) are
phylogenetically (Fig. 2) and morphologically (Fig. 3) diverse. Most of the FMR grow
by oxidizing organic compounds or hydrogen with the reduction of Fe(III) or Mn(IV),
but S° oxidation coupled to Fe(III) reduction also can provide energy to support
growth of microorganisms growing at low pH. The various types of FMR are briefly
described below.
Fig. 2. Phylogenetic tree, based on 16S rDNA sequences, of microorganisms known
to conserve energy to support growth from Fe(III) reduction. The tree was inferred
using the Kimura two-parameter model in TREECON for Windows (Van der Peer and
De Wachter, 1994). Bootstrap values at nodes were calculated from one hundred
replicates.
Fig. 3. Phase contrast micrographs of various organisms that conserve energy to
support growth from Fe(III) reduction. Bar equals 5 μm, all micrographs at
equivalent magnification.
Table 2. Organisms known to conserve energy to support growth from Fe(III)
reduction.
Geobacteraceae
Most of the known FMR, available in pure culture, that can oxidize organic
compounds completely to carbon dioxide with Fe(III) or Mn(IV) serving as the sole

electron acceptor are in the family Geobacteraceae in the delta δ-Proteobacteria (Fig.
2; Table 2). The family Geobacteraceae is comprised of the genera Geobacter,
Desulfuromonas , Desulfuromusa and Pelobacter. With the exception of the
Pelobacter species, all of the Geobacteraceae genera contain microorganisms that
oxidize acetate to carbon dioxide. This metabolism is significant because, as
discussed above, acetate is probably the primary electron donor for Fe(III) reduction
in most sedimentary environments. Many of these Geobacteraceae also can use
hydrogen as an electron donor for Fe(III) reduction. Various species in the
Geobacteraceae oxidize a variety of other organic acids, including in some instances
long-chain fatty acids (Table 2). Several species of Geobacter have the ability to
anaerobically oxidize aromatic compounds, including the hydrocarbon toluene.
Geobacteraceae are the Fe(III) reducers most commonly recovered from a variety of
sedimentary environments when the culture media contains acetate as the electron
donor and Fe(III) oxide or the humic acid analog, anthraquinone-2,6-disulfonate
(AQDS) as the electron acceptor (Coates et al., 1996; Coates et al., 1998).
Furthermore, analysis of 16S rDNA sequences in sandy aquifer sediments in which
Fe(III) reduction was the predominant terminal electron accepting process indicated
that Geobacter species were a major component of the microbial community
(Rooney-Varga et al., 1999; Synoeyenbos-West et al., 1999).
Geothrix
Geothrix fermentans and closely related strains have been recovered from the
Fe(III)-reducing zone of petroleum-contaminated aquifers (Anderson et al., 1998;
Coates et al., 1999b). Like Geobacter species, G. fermentans can oxidize short-chain
fatty acids to carbon dioxide with Fe(III) serving as the sole electron acceptor. It can
also use long-chain fatty acids as well hydrogen as an electron donor for Fe(III)
reduction (Table 2) and can grow fermentatively on several organic acids. G.
fermentans, along with Holophaga foetida, is part of a deeply branching group in the
kingdom Acidobacterium. The 16S rDNA sequences from this kingdom are among the
most common recovered from soil, but few organisms from this kingdom have been
cultured (Barns et al., 1999). Studies in which Fe(III)-reducing microorganisms were

recovered in culture media suggested that organisms closely related to G.
fermentans might be as numerous as Geobacter species in the Fe(III) reduction zone
of a petroleum-contaminated aquifer (Anderson et al., 1998). However, analyses of
16S rDNA sequences have indicated that Geothrix sp. are probably several orders of
magnitude less numerous than Geobacter species in such environments (Rooney-
Varga et al., 1999; Synoeyenbos-West et al., 1999).
Geovibrio ferrireducens and Deferribacter thermophilus
Culturing from hydrocarbon-impacted soils and a petroleum reservoir have led to the
recovery of the mesophile, Geovibrio ferrireducens (Caccavo et al., 1996) and the
thermophile, Deferribacter thermophilus (Greene et al., 1997). These organisms are
more closely related to each other than to any other known Fe(III)-reducing
microorganisms and grow with similar electron donors for Fe(III)-reduction. G.
ferrireducens has been shown to completely oxidize its carbon substrates to carbon
dioxide and it is assumed that D. thermophilus can as well, but this has not been
directly tested. An interesting feature of the metabolism of these organisms is the
ability to use some amino acids as electron donors for Fe(III) reduction. The
environmental distribution of these organisms has not been studied in detail.
Ferribacter limneticum
Ferribacter limneticum (Cummings et al., 1999) is the only organism in the β-
subclass of the Proteobacteria that is known to conserve energy to support growth
from Fe(III) reduction. Unlike many Fe(III)-reducing microorganisms it does not
utilize Mn(IV) as an electron acceptor. To date, this organism has only been
recovered from mining-impacted lake sediments.
Shewanella–Ferrimonas–Aeromonas
In contrast to the organisms discussed above, which only grow anaerobically, several
genera within the γ-Proteobacteria, can grow aerobically, and under anaerobic
conditions can use Fe(III), Mn(IV), or other electron acceptors (Table 2). These
include species of Shewanella, Ferrimonas, and Aeromonas. Although many of these
organisms can use a wide range of electron donors when oxygen is available as an
electron acceptor, their range of electron donors with Fe(III) and Mn(IV) is generally

restricted to hydrogen and small organic acids. An exception is Shewanella
saccharophila, which also can use glucose as an electron donor for Fe(III) reduction.
The Shewanella species, which have been studied in detail, incompletely oxidize
multicarbon organic electron donors to acetate.
Another Fe(III)-reducing microorganism that may be related to this group is an
unidentified microorganism referred to as a "pseudomonad," which was the first
organism found to grow with hydrogen as the electron donor and Fe(III) as the
electron acceptor (Balashova and Zavarzin, 1980). However, this organism does not
appear to be available in culture collections for further study, and its true
phylogenetic placement is unknown.
The FMR in the γ-Proteobacteria have been recovered from a variety of sedimentary
environments including various aquatic sediments (Myers and Nealson, 1988;
Caccavo et al., 1992; Coates et al., 1999a) and the subsurface (Pedersen et al.,
1996; Fredrickson et al., 1998). However, in contrast to the organisms in the
Geobacteraceae which are found to be numerous in both molecular and culturing
analysis of widely diverse environments where Fe(III) reduction is important, the
distribution of Shewanella is more variable. For example, Shewanella were found to
account for ca. 2% of the microbial population in some surficial aquatic sediments,
but could not be detected in other sediments (DiChristina and DeLong, 1993).
Shewanella 16S rDNA sequences could not be recovered from aquifer sediments in
which Fe(III) reduction was the predominant terminal electron-accepting process
TEAP (Synoeyenbos-West et al., 1999). This was the case even when electron
donors, such as lactate and formate, that are preferred by Shewanella species, were
added to stimulate Fe(III) reduction.
Sulfurospirillum barnesii
Sulfurospirillum barnesii which was initially isolated based on its ability to use
selenate as an electron acceptor (Oremland et al., 1994b), also can grow using the
reduction of Fe(III) and the metalloid As(V) (Laverman et al., 1995). Although it has
commonly been found that if one organism in a close phylogenetic group has the
ability to reduce Fe(III) then others in the group also will be Fe(III) reducers (Roden

and Lovley, 1993a; Lovley et al., 1995c; Lonergan et al., 1996; Kashefi and Lovley,
1999), Sulfurospirillum arsenophilum does not reduce iron [Fe(III); Stolz et al.,
1999)]. Wolinella succinogenes, which is also in the ε-subclass of the Proteobacteria,
also can reduce Fe(III) and metalloids (Lovley et al., 1997c; 1999b), but whether W.
succinogenes conserves energy to support growth from metal reduction has not been
determined.
Acidophilic Fe(III)- reducing Microorganisms
Although Fe(III) is highly insoluble at the circumneutral pH at which most Fe(III)-
reducing microorganisms have been studied, Fe(III) is soluble at low pH. The redox
potential of the Fe
+3
/Fe
+2
redox couple is significantly more positive than the Fe(III)
oxide/Fe
+2
redox couple and the oxidation of electron donors (such as S°) that might
be unfavorable at circumneutral pH with Fe(III) oxides as the electron acceptors
might be favorable in acidic pH where more Fe
+3
is available. Thiobacillus ferroxidans
can grow anaerobically with S° as the electron donor and Fe(III) as the electron
acceptor (Das et al., 1992; Pronk et al., 1992). Thiobacillus thiooxidans also has
been shown to reduce Fe(III) with S° as the electron donor (Brock and Gustafson,
1976), but the culture was grown aerobically and energy conservation from Fe(III)
reduction was not demonstrated. This was also true of the thermophile, Sulfolobus
acidocaldarius (Brock and Gustafson, 1976).
Acidophilic thermophiles that can reduce Fe(III) with glycerol or thiosulfate as the
electron donor have been described (Bridge and Johnson, 1998), but the ability of
these organisms to conserve energy to support growth from Fe(III) reduction has

not been examined in detail. An acidophilic mesophile, designated strain SJH,
exhibited Fe(III)-dependent growth in a complex organic medium containing glucose
and tryptone (Johnson and McGinness, 1991), but further characterization of the
electron donors for Fe(III) reduction and a detailed description of the organism were
not provided.
Hyperthermophilic and Thermophilic Archaea and Bacteria
In addition to D. thermophilus mentioned above, a number of other thermophiles
and hyperthermophiles can conserve energy to support growth from Fe(III)
reduction. The first thermophilic FMR reported was the deep subsurface isolate,
Bacillus infernus, which has a temperature optimum of 60°C (Boone et al., 1995). It
was also the first Gram-positive FMR identified. In contrast to all other members of
the Bacillus genus, B. infernus is a strict anaerobe and can grow by fermentation
when Fe(III) or other electron acceptors are not available. Other thermophilic FMR
recovered from subsurface environments include Thermoterrabacterium
ferrireducens (Slobodkin et al., 1997) and a Thermus species (Kieft et al., 1999).
As summarized in Tables 1 and 2, a wide phylogenetic diversity of hyperthermophilic
microorganisms can transfer electrons to iron [Fe(III); Vargas et al., 1998)].
However, only three of these organisms, Pyrobaculum islandicum , P. aerophilum,
and Thermotoga maritima, have been shown to conserve energy to support growth
from Fe(III) reduction. P. islandicum and T. maritima grow with hydrogen as the
electron donor and Fe(III) as the electron acceptor and P. islandicum and P.
aerophilum also can grow with complex organic matter (peptone, yeast extract) as
the electron donor and Fe(III) as the electron acceptor (Kashefi and Lovley, 1999).
Forms of Fe(III) and Mn(IV) That Can Serve as Electron Acceptors
Unlike other types of respiration that use soluble electron acceptors, Fe(III) and
Mn(IV) reduction require the reduction of insoluble electron acceptors in most
environments. The insoluble Fe(III) and Mn(IV) oxides that are the most
environmentally relevant forms of Fe(III) and Mn(IV) at circumneutral pH can be
found in a wide diversity of forms (Dixon and Skinner, 1992; Schwertmann and
Fitzpatrick, 1992). The nature of the oxides have a major impact on the rate and

extent of Fe(III) and Mn(IV) reduction (Lovley, 1991a; Lovley, 1995a).
Pure cultures of Fe(III)-reducing microorganisms reduce a variety of insoluble Fe(III)
and Mn(IV) forms (Lovley, 1991a), including the Fe(III) oxides naturally found in
sedimentary environments (Lovley et al., 1990b; Coates et al., 1996). Early studies
on Fe(III) reduction by fermentative microorganisms often employed highly
crystalline Fe(III) oxides as the Fe(III) form (Table 1). However, studies on Fe(III)
reduction in sediments suggested that the primary form of Fe(III) that FMR reduced
in aquatic sediments was poorly crystalline Fe(III) oxides and that poorly crystalline
Fe(III) oxides promoted the complete oxidation of organic compounds to carbon
dioxide with Fe(III) serving as the electron acceptor (Lovley and Phillips, 1986a;
Lovley and Phillips, 1986b; Phillips et al., 1993).
The use of poorly crystalline Fe(III)-oxide as the Fe(III) form permitted the first
recovery of a microorganism that could completely oxidize organic compounds to
carbon dioxide with Fe(III) serving as the electron acceptor (Lovley et al., 1987c).
Most subsequent studies that have enriched for Fe(III)-reducing microorganisms
from the environment or that have evaluated mechanisms for Fe(III) oxide reduction
by pure cultures of FMR have used poorly crystalline Fe(III) oxide as the electron
acceptor.
FMR have been shown to reduce some of the more crystalline Fe(III) oxides,
including hematite, goethite, akaganeite, and magnetite, under some conditions
(Table 2; Lovley, 1991a; Kostka and Nealson, 1995; Roden and Zachara, 1996).
However, the rates of reduction of the crystalline Fe(III) oxides are generally much
slower than the reduction of poorly crystalline Fe(III) oxide. In most instances,
sustained growth is difficult to maintain in consecutive transfer of pure cultures with
crystalline Fe(III) oxides as the electron acceptor. In evaluating the potential for
reduction of crystalline Fe(III) oxides, it is important to omit complex organic matter
or organic acids, which chelate and solubilize Fe(III) from the Fe(III) oxides. The
FMR reduction of crystalline Fe(III) oxides in soils and sediments has not been
demonstrated conclusively.
An alternative, environmentally relevant, source of insoluble Fe(III) is structural

Fe(III) in clays. Reduction of Fe(III) in clays is often observed in flooded soils and
FMR have been shown to reduce this iron [Fe(III); Kostka et al., 1996; Lovley et al.,
1998)].
Soluble Fe(III) forms are often used for culturing FMR. Although soluble Fe(III) may
not represent an environmentally significant form of Fe(III), it provides an easy
method for culturing FMR. Pure cultures generally reduce soluble Fe(III) forms faster
than poorly crystalline Fe(III) oxide, and less insoluble precipitates are formed
during reduction of soluble Fe(III). Furthermore, unlike poorly crystalline Fe(III)
oxide, some soluble Fe(III) forms do not have to be synthesized because they are
commercially available.
Fe(III)-citrate is the most commonly used form of soluble Fe(III) for the culture of
FMR. It is highly soluble and can readily be provided at concentrations as high as 50
mM, even in media with a high salt content. However, Fe(III)-citrate may be toxic to
some Fe(III)-reducing microorganisms (Lovley et al., 1990a; Lovley et al., 1993b;
Roden and Lovley, 1993b). The Fe(III) chelated with nitrilotriacetic acid (Fe(III)-
NTA) is a useful alternative. The limitations of Fe(III)-NTA are its frequent toxicity at
concentrations above 10 mM and its tendency to precipitate as Fe(III) oxide when
Fe(III)-NTA is added to media with high salt content or at temperatures of 60°C or
above. Unlike Fe(III)-citrate, Fe(III)-NTA is not commercially available and must be
synthesized, as described below. "Ferric pyrophosphate" has been successfully used
for the culture of FMR (Caccavo et al., 1994; Caccavo et al., 1996). This is a
somewhat undefined mixture that contains not only Fe(III) and phosphate, but also
citrate and nitrilotriacetic acid which are likely to play an important role in
maintaining the solubility of Fe(III) in this mixture.
The most commonly used form of Mn(IV) oxide in studies of Mn(IV) reduction by
FMR is birsnessite, a readily synthesized Mn(IV) oxide (see method for synthesis
below). However, there is a wide diversity of Mn(IV) oxides is found in the
environment and rates of Mn(IV) reduction can be dependent upon the form of
Mn(IV) oxide available (Burdige et al., 1992).
Products of Fe(III) and Mn(IV) Reduction

Products Fe(II) and Mn(II) are more soluble than Fe(III) and Mn(IV) and thus
microbial Fe(III) and Mn(IV) reduction results in a marked increase in dissolved iron
and manganese in anaerobic environments and in cultures of FMR. However, in both
cultures and sediments, most of the Fe(II) and Mn(II) produced during microbial
reduction of insoluble Fe(III) and Mn(IV) oxides often remains in solid forms (Lovley,
1991a; Lovley, 1995a; Schnell et al., 1998). In culture, microbial Fe(III) and Mn(IV)
reduction has been shown to form such minerals as magnetite (Fe
3
O
4
) siderite
(FeCO
3
), vivianite (Fe
3
PO
4
· 8H
2
O) and rhodochrosite (MnCO
3
; Lovley, 1991a; Lovley,
1995b). The formation of such minerals in culture provides a model for the
geologically significant deposition of iron and manganese minerals described above.
The fact that most of the Fe(II) and Mn(II) produced from microbial Fe(III) and
Mn(IV) reduction is insoluble means that quantitative analysis of Fe(III) or Mn(IV)
reduction either in cultures or environmental samples requires quantifying the
amount of insoluble Fe(II) or Mn(II) produced. The Fe(II) may be solubilized in HCl
(Lovley and Phillips, 1986a) or oxalate (Phillips and Lovley, 1987; Lovley and Phillips,
1988c) before measurement with Fe(II)-specific reagents such as ferrozine (Stookey,

1970) or ion chromatography (Schnell et al., 1998). Loss of Fe(III) in acid-solubilized
samples also can be monitored (Lovley and Phillips, 1988b; Schnell et al., 1998).
Methods for quantitatively measuring Mn(IV) reduction are not as well established.
Much of the Mn(II) produced during Mn(IV) reduction adsorbs onto the Mn(IV) oxide
or forms insoluble Mn(II) minerals. Mn(II) can be solubilized in acid and soluble
manganese measured with atomic absorption spectroscopy (Lovley and Phillips,
1988c), but this is technically difficult because acid will also eventually dissolve the
Mn(IV) oxide. A better strategy might be to solubilize all the manganese and
specifically measure the Mn(II) produced with ion chromatography (Schnell et al.,
1998).
Mechanisms for Electron Transfer to Fe(III) and Mn(IV)
The mechanisms by which Fe(III)- and Mn(IV)-reducing microorganisms transfer
electrons to insoluble Fe(III) and Mn(IV) are poorly understood. It is generally stated
that Fe(III) and Mn(IV) reducers must directly reduce Fe(III) and Mn(IV) oxides by
establishing contact with the oxides (Lovley, 1991a). Until recently, the primary
evidence of the need for contact was the finding that Fe(III) and Mn(IV) were not
reduced when Fe(III) or Mn(IV) oxides and Fe(III)- and Mn(IV)-reducing
microorganisms were separated by semipermeable membranes, which should permit
the passage of soluble substances. This result as well was considered evidence that
Fe(III)- and Mn(IV)-reducing microorganisms do not produce chelators to solubilize
Fe(III) or Mn(IV) and do not produce compounds that could serve as soluble
electron-shuttles between Fe(III)- and Mn(IV)-reducing microorganisms and the
insoluble oxides. However, recent studies have demonstrated that this approach is
flawed because even when chelators or electron shuttles were added to cultures,
Fe(III)-reducing microorganisms still did not significantly reduce Fe(III) oxide held
within dialysis tubing (Nevin and Lovley, 1999a). Studies with strains of Shewanella
alga, which were deficient in the ability to attach to Fe(III) oxides, continued to
reduce Fe(III), suggesting that attachment to Fe(III) oxide was not necessary for
Fe(III) oxide reduction (Caccavo et al., 1997). Thus, although studies have
documented the association of Fe(III)-reducing microorganisms with Fe(III)-oxide

particles, the current evidence is not definitive to clearly state that Fe(III)- and
Mn(IV)-reducing microorganisms must attach to Fe(III) and Mn(IV) oxides in order
to reduce them.
It was suggested that Geobacter sulfurreducens might reduce Fe(III) oxide in culture
by releasing a low molecular weight (9.6 kDa) c-type cytochrome into the medium
which could serve as a soluble electron shuttle between G. sulfurreducens and the
Fe(III) oxide (Seeliger et al., 1998). However, further investigation has
demonstrated that this c-type cytochrome is not an effective electron shuttle and
that in healthy, actively growing cultures of G. sulfurreducens , little, if any, of the
9.6 kDa cytochrome is released into the growth medium (Lloyd et al., 1999).
Therefore, the proposed shuttling mechanism is unlikely.
Iron [Fe(III)]-reducing microorganisms can use humics and other extracellular
quinones as electron shuttles to promote Fe(III) oxide reduction (Lovley et al., 1996;
Lovley et al., 1998; Lovley et al. 2000). As discussed below, humics and other
extracellular quinones can serve as electron acceptors for Fe(III)-reducing
microorganisms. The hydroquinone moieties that are generated as the result of the
reduction of extracellular quinones can transfer electrons to Fe(III) oxides through a
strictly abiotic reaction. This reduction of Fe(III) regenerates quinone moieties that
can then again serve as electron acceptors for Fe(III)-reducing microorganisms. In
this manner a small amount of extracellular quinone can promote a significant
increase in the rate of reduction of poorly crystalline Fe(III) oxide. For example,
studies with cultures and aquifer sediments have demonstrated that there is a
significant potential for electron shuttling with as little as 100 nM AQDS (Lloyd et al.,
1999; Nevin and Lovley, 1999b). Although electron shuttling to Mn(IV) oxides have
not been studied in detail, a similar phenomenon is expected.
However, both the evidence that Fe(III)- and Mn(IV)-reducing microorganisms can
reduce Fe(III) and Mn(IV) oxides in cultures without added electron shuttling
compounds and chelators and the lack of evidence for release of electron shuttling or
chelating compounds by the microorganisms (Nevin and Lovley, 1999a) suggests
that FMR can directly transfer electrons to Fe(III) and Mn(IV) oxides. The Fe(III)-

reductase activity is primarily localized in the membranes of Fe(III)- and Mn(IV)-
reducing microorganisms such as G. metallireducens (Gorby and Lovley, 1991), S.
putrefaciens (Myers and Myers, 1993), and G. sulfurreducens (Gaspard et al., 1998;
Magnuson et al., 1999). The involvement of cytochromes of the c-type has been
suggested to be involved in electron transport to Fe(III) in G. metallireducens
(Lovley et al., 1993c) and S. putrefaciens (Myers and Myers, 1992; Myers and
Myers, 1997; Beliaev and Saffarini, 1998). A NADH-dependent Fe(III) reductase
complex was purified from G. sulfurreducens and a 90-kDa c-type cytochrome in the
complex served as the Fe(III) reductase (Magnuson et al., 1999). However, no study
has as yet definitively identified as yet the physiologically relevant Fe(III) or Mn(IV)
reductase in any organism capable of conserving energy to support growth via
Fe(III) or Mn(IV) reduction.
Other Respiratory Capabilities of FMR
Many FMR can reduce other electron acceptors well-known to support anaerobic
respiration such as fumarate, nitrate, and S° (Table 2). Fumarate is reduced to
succinate, and S° is reduced to sulfide. In those documented instances of nitrate
reduction, nitrite or ammonia has been found to be the product. It is interesting that
nearly all microorganisms with the ability to reduce Fe(III) also can reduce S° to
sulfide. In fact, screening of known S°-reducing microorganisms already available in
culture has been a fruitful approach for discovering new FMR (Roden and Lovley,
1993a; Lonergan et al., 1996; Vargas et al., 1998).
Electron Transfer to Other Metals and Metalloids
Many Fe(III)-reducing microorganisms can transfer electrons to metals other than
iron or manganese [Fe(III) or Mn(IV); Table 2]. For example, G. metallireducens and
S. putrefaciens can grow with U(VI) as the sole electron acceptor (Lovley et al.,
1991b). Cell suspensions of other FMR have been found to transfer electrons to
U(VI), but their ability to obtain energy to support growth from U(VI) reduction has
not been evaluated. Many sulfate-reducing microorganisms, can effectively reduce
U(VI), but attempts to grow these organisms with U(VI) as the sole electron acceptor
have been unsuccessful (Lovley et al., 1993b).

U(VI), which is soluble in bicarbonate-based media is reduced to U(IV) that
precipitates as the mineral uraninite (Gorby and Lovley, 1992; Lovley and Phillips,
1992). Visualization of microbial U(VI) reduction can be enhanced with the use a
fluorescent light. The U(VI)-containing liquid cultures or agar plates fluoresce green,
whereas the uraninite does not significantly fluoresce. Loss of U(VI) during U(VI)
reduction can be monitored as loss of soluble uranium by monitoring total uranium
concentrations in culture filtrates, but since U(IV) precipitation is not instantaneous
(Gorby and Lovley, 1992), more quantitative estimates of U(VI) reduction can be
more quantitatively estimated by monitoring loss of U(VI) with a kinetic
phosphorescence analyzer (Lovley et al., 1991b) or by using ion chromatography.
Several Fe(III)-reducing microorganisms can reduce the oxidized form of the
radioactive metal technetium, Tc(VII) to reduced forms (Table 2). Growth with
Tc(VII) as the sole electron acceptor has not yet been documented as yet in any
organism. Tc(VII) reduction can be monitored by following the formation of reduced
technetium forms with paper chromatography and a phosphorimager (Lloyd and
Macaskie, 1996).
FMR can reduce a variety of other metals and metalloids (Table 2). Several can
reduce Cr(VI) to Cr(III), but growth with Cr(VI) as the sole electron acceptor has not
been demonstrated (Lovley, 1995c). The FMR, S. barnesii can conserve energy from
the reduction of Se(VI) to Se° and As(V) to As(III) (Laverman et al., 1995).
Electron Transfer to and from Humic Substances and Other Extracellular
Quinones
All FMR that have been evaluated to date, including the hyperthermophiles, have the
ability to transfer electrons to humic substances (humics) or other extracellular
quinones such as the humics analog, anthraquinone-2,6-disulfonate (AQDS) Lovley
et al., 1996; Lovley et al., 1998; Lovley et al. 2000). In those organisms in which
the potential for growth has been evaluated, energy to support growth is from
electron transport to humics and this capability is conserved. Electron-spin
resonance (ESR) studies have suggested that quinones are important electron-
accepting groups in the humics (Scott et al., 1998). The ESR studies with AQDS as

the sole electron acceptor have directly demonstrated that energy can be conserved
from electron transfer to extracellular quinones has been directly demonstrated in
studies with AQDS as the sole electron acceptor (Lovley et al., 1996; Coates et al.,
1998; Lovley et al., 1998). Humics can chelate Fe(III) that is also available for
microbial reduction (Benz et al., 1998; Lovley and Blunt-Harris, 1999a), but the
concentration of microbially reducible Fe(III) in humics is a minor fraction of the total
electron-accepting capacity (Lovley and Blunt-Harris, 1999a).
A wide diversity of humics can serve as electron acceptors for Fe(III)-reducing
microorganisms (Lovley et al., 1996; Scott et al., 1998). Highly purified reference
humics that have been extracted from diverse environments can be obtained from
the International Humic Substances Society. Other commercially available humics
are highly impure, differ from humics found in soils and sediments, and therefore
should be avoided for definitive studies because commercially available humics are
highly impure and their characteristics are unlike the humics found in soils and
sediments (Malcolm and MacCarthy, 1986).
The expense and technical difficulty of conducting studies with humics makes it
preferable to carry out some studies on microbial reduction of extracellular quinones
with humics analogs, such as AQDS (Lovley et al., 1996; Lovley et al., 1998). The
advantages of AQDS are its low cost, high solubility, and its easy detection [an
orange color develops when AQDS is reduced to anthrahydroquinone-2,6-disulfonate
(AHQDS)].
Several FMR have the ability to use reduced extracellular quinones as an electron
donor for reduction of electron acceptors such as nitrate and fumarate (Lovley et al.,
1999b). Shewanella alga and Geobacter sulfurreducens grew with AHQDS as the
electron donor. However, other FMR that could oxidize AHQDS in cell suspensions
could not be grown with AHQDS as the sole electron acceptor. The ability of FMR to
both reduce and oxidize extracellular quinones permits their use with other quinone-
oxidizing and quinone-reducing microorganisms as an interspecies electron transfer
system in which quinones serve as the electron shuttle between the microorganisms
(Lovley et al., 1999b).

Proton Reduction in Syntrophic Association with Hydrogen-consuming
Microorganisms
In the absence of Fe(III) or other suitable electron acceptors, some organisms in the
Geobacteraceae can transfer electrons to protons to produce hydrogen gas. For
hydrogen production to be thermodynamically favorable, a sink for hydrogen, such
as a hydrogen-consuming microorganism, must keep hydrogen concentrations low
enough. For example, several Pelobacter species can oxidize ethanol to acetate and
carbon dioxide when grown in association with hydrogen-consuming microorganisms
(Schink, 1992). G. sulfurreducens can oxidize acetate to carbon dioxide when
cultured with Wolinella succinogenes, which oxidizes hydrogen with concomitant
reduction of nitrate (Cord-Ruwisch et al., 1998).
Reductive Dechlorination
Several Fe(III)-reducing microorganisms are capable of using chlorinated compounds
as electron acceptors. Desulfuromonas chlorethenica, which was isolated as a
tetrachloroethylene-respiring microorganism (Krumholz et al., 1996; Krumholz,
1997) was found to grow also with Fe(III) as the electron acceptor, as expected for
microorganisms within the family Geobacteraceae (Lonergan et al., 1996). Other
Geobacteraceae that were evaluated did not reduce tetrachloroethylene.
Desulfitobacterium dehalogenans which can use chlorophenolic compounds as an
electron acceptor (Utkin et al., 1994), also can grow with Fe(III) as the electron
acceptor (Lovley et al., 1998). Another chlorophenol-respiring species in the same
genus, Desulfitobacterium hafniense, was reported to reduce Fe(III), but it was not
reported whether growth was conserved from Fe(III) reduction (Christiansen and
Ahring, 1996).
Recovery of Fe(III)- and Mn(IV)- reducing
Microorganisms in Culture
Localizing Zones of Fe(III) and Mn(IV) Reduction
Although FMR can be recovered from nearly any soil or sediment sample, it is
generally of interest to study organisms from habitats in which Fe(III) and Mn(IV)
are ongoing processes. Dissimilatory Fe(III) and Mn(IV) reduction are geochemically

most significant in anaerobic environments such as freshwater and marine
sediments; flooded soils or the anaerobic interior of soil aggregates; the deep
terrestrial subsurface; and shallow aquifers contaminated with organic compounds.
In aquatic sediments and the terrestrial subsurface Fe(III) and Mn(IV) reduction are
most apparent in discrete anoxic sediment layers in which the endproducts of Fe(III)
and Mn(IV) reduction, Fe(II) or Mn(II), are accumulating. In the typical zonation of
respiratory processes found with depth in aquatic sediments or along the
groundwater flow path in the subsurface, the zones of Fe(III) and Mn(IV) reduction
are typically bounded on one side by the zone of nitrate reduction and on the other
side by the zone of sulfate reduction (Lovley and Chapelle, 1995c). In addition to
these larger discrete zones of Fe(III) reduction and Mn(IV) reduction in sedimentary
environments, it is important to recognize that many soils and sediments that are
predominately aerobic alsomay contain abundant anaerobic microzones in which
Fe(III) and Mn(IV) reduction may be taking place.
Although accumulation of dissolved Fe(II) and Mn(II) in groundwater or porewater
can be used to help identify the zones of Fe(III) and Mn(IV) reduction in subsurface
or aquatic sediments, such standard geochemical measurements can often fail to
accurately locate the metal reduction zones (Lovley et al., 1994b). A primary reason
for this failure is that high concentrations of Fe(II) and Mn(II) may be found in
sediments in which other TEAPs, such as methanogenesis, predominate.
In environments where conditions approach steady-state such as aquatic sediments
and aquifers, in which conditions approach steady-state, measurements of dissolved
hydrogen can be used to identify zones in which Fe(III) reduction is the TEAP (Lovley
and Goodwin, 1988a; Lovley et al., 1994c). This is because there is a unique range
of dissolved hydrogen that is associated with Fe(III) reduction that is the
predominant TEAP in steady-state environments. Hydrogen measurements have not
been used to localize Mn(IV)-reducing zones because: 1) hydrogen concentrations
under Mn(IV)-reducing conditions are very low and difficult to accurately measure
accurately; 2) hydrogen concentrations for Mn(IV) and nitrate reduction are similar;
and 3) the low concentrations of Mn(IV) in many soils means that the Mn(IV)

reduction zone is not extensive.
An alternative method for determining the zone of Fe(III) reduction in soils and
sediments is to use [2-
14
C]-acetate (Lovley, 1997a). The reduction of Fe(III) can be
considered to be the TEAP if: 1) a tracer quantity of [2-
14
C]-acetate added to the
sediments is converted to
14
CO
2
with no production of
14
CH
4
; 2) the production of
14
CO
2
is not inhibited with the addition of molybdate; 3) the sediments are depleted of
nitrate; and 4) the sediments contain some Fe(II). The reasoning for this is that: 1)
lack of
14
CH
4
production rules out methanogenesis as a TEAP; 2) molybdate inhibits
acetate oxidation by sulfate reducers so the lack of inhibition with molybdate rules
out sulfate reduction as the TEAP; 3) nitrate reduction can not be an important TEAP
in the absence of nitrate; and 4) Mn(IV) reduction can not be the TEAP in the

presence of Fe(II) because Fe(II) rapidly reacts with Mn(IV) (Lovley and Phillips,
1988b) and thus Fe(II) will only be found if reactive Mn(IV) has been depleted.
The rates of other TEAPs can often be quantified in sediments with the use of
radiotracers. Unfortunately, attempts to measure rates of Fe(III) reduction in
sediments with radioactively labeled Fe(III) were unsuccessful (Roden and Lovley,
1993b). This was because there was rapid isotope exchange between the
radiolabelled Fe(III) and other iron pools, including Fe(II), was rapid. Thus, it was
not possible to monitor rates of microbial Fe(III) reduction by measuring the
production of radiolabeled Fe(II) from labeled Fe(III).
Rates of Fe(III) and Mn(IV) reduction in sediments can be estimated from anaerobic
incubations of sediments by monitoring the accumulation of Fe(II) and Mn(II) are
monitored over time. It is important that the solid phase Fe(II) and Mn(II) pools be
measured after acidic extractions or some other technique because most of the
Fe(II) and Mn(II) are not recovered in the dissolved phase (Lovley and Phillips,
1988c; Lovley, 1991a). Geochemical modeling has been used to estimate rates of
Fe(III) and Mn(IV) reduction in some aquatic sediments and subsurface
environments and potentially could be used to identify zones of Fe(III) and Mn(IV)
reduction (Lovley, 1995a).
Isolation Procedures
Although some FMR also can use oxygen as an electron acceptor or are tolerant of
exposure to air, many are strict anaerobes. Therefore, unless the goal is to
specifically select for facultative FMR, the use of strict anaerobic technique is
preferable in initial enrichment and/or isolation procedures. To date, most FMR have
been recovered using slight modifications of standard (Miller and Wolin, 1974; Balch
et al., 1979) anaerobic techniques. This involves the use of culture tubes or bottles
fitted with thick butyl rubber stoppers; removing traces of oxygen from gases by
passing the gases through a column of heated copper filings; and carrying out
transfers with syringes and needles or under a stream of anoxic gas.
Culture media can be prepared with the classical approach (Hungate, 1969) of
boiling the media under a stream of anoxic gas to remove dissolved oxygen and then

dispensing into tubes or bottles under anaerobic conditions. Alternatively, aerobic
media may be dispensed into individual tubes or bottles and then the media can be
vigorously bubbled with anoxic gas to strip dissolved oxygen from the media (Lovley
and Phillips, 1988c). Both media preparation approaches appear to yield similar
organisms. Reducing agents such as Fe(II)—typically supplied at 1–3 mM as ferrous
chloride—cysteine (0.25–1 mM), or sulfide (0.25–1 mM) can be added to dispensed
media from anoxic stocks just prior to inoculation. In addition to reacting with any
trace oxygen in the media, cysteine and sulfide will reduce Fe(III) and Mn(IV) in the
media, producing Fe(II) and Mn(II). Fe(II) rapidly reacts with traces of oxygen,
forming Fe(III). Manganese [Mn(II)] will only slowly react abiotically with oxygen.
Many FMR have been recovered without the addition of reducing agents to the
media. Once Fe(III) reduction begins, the Fe(II) formed serves as protection against
oxygen contamination. Reducing agents are rarely used in media designed for liquid-
to-liquid transfer of Fe(III)-reducing cultures because the inoculum of the Fe(III)-
reducing cultures typically contain millimolar quantities of dissolved Fe(II), which will
scavenge traces of oxygen from the media to which the inoculum has been added.
A variety of media has been successfully employed for the enrichment and isolation
of FMR, many of which are given in the references provided with each of the
organisms in Table 2. An example of a freshwater and a marine medium are
provided below. No definitive comparative studies of the efficacy of various media in
recovering FMR have been carried out. However, it has been found that the
freshwater medium described here can be used to recover Geobacter species with
16S rDNA sequences that are closely related to the 16S rDNA sequences that
predominate in the Fe(III) reduction zone of sandy aquifers (Rooney-Varga et al.,
1999; Synoeyenbos-West et al., 1999).
Most successful isolations of pure cultures of Fe(III)- and Mn(IV)-reducing
microorganisms have used either organic acids, primarily acetate or lactate, or
hydrogen as the electron donor. If an enrichment step is used in the initial stages of
recovery of the organisms, then fermentable compounds such as glucose generally
result in the enrichment of fermentative microorganisms. However, as summarized

above, some Fe(III)- and Mn(IV)-reducing microorganisms can use sugars and
amino acids as electron donors and these electron donors potentially could be be
used for direct isolation of FMR.
A variety of Fe(III) and Mn(IV) forms that were discussed above can be used as
electron acceptors for enrichment or isolation. Iron added as Fe(III)-citrate and
Fe(III) pyrophosphate is not ideal for enrichment cultures as the citrate is rapidly
degraded by microorganisms other than Fe(III) reducers. Once the citrate is
degraded, the Fe(III) from the Fe(III)-citrate precipitates as an insoluble Fe(III)
oxide and thus defeats the purpose of adding the chelator. The compound Fe(III)-
NTA is relatively resistant to anaerobic degradation and can be used as a soluble
source of Fe(III) for enrichment of Fe(III) reducers. However, as noted above, it is
not suitable for use in media with marine salinities or at high temperature. Both
Fe(III)-citrate and Fe(III)-NTA are toxic to some Fe(III) reducers. Although
solubilization of Mn(IV) with various chelators for use in recovery of Mn(IV)-reducing
microorganisms may be possible, this approach has not been widely used.
As noted above, poorly crystalline Fe(III) oxide is typically the insoluble Fe(III) oxide
of choice for culturing. A wide diversity of other Fe(III) oxides can be synthesized
(Schwertmann and Cornell, 1991), if desired. If the media is dispensed aerobically
into culture vessels, then a slurry of the Fe(III) or Mn(IV) oxide can be added to the
vessels prior to addition of the media. An advantage of using poorly crystalline
Fe(III) oxide as the electron acceptor is that most Fe(III)-reducing microorganisms
convert the poorly crystalline Fe(III) oxide to the magnetic mineral magnetite during
reduction. This is visually apparent as the reddish, non-magnetic Fe(III) oxide is
transformed into a black, highly magnetic precipitate (Lovley et al., 1987c).
Reduction of the Mn(IV) oxide is also visually apparent in bicarbonate-buffered media
because reduction of the dark Mn(IV) oxide results in its dissolution and concomitant
accumulation of rhodochrosite, a white Mn(II) carbonate mineral.
An alternative electron acceptor that can be used for the recovery of Fe(III)- and
Mn(IV)-reducing microorganisms is the humics analog, AQDS, which is typically
provided at 5 mM. All of the Fe(III)-reducing microorganisms that have been

evaluated can reduce AQDS, whereas microorganisms that do not reduce Fe(III) can
not reduce AQDS (Lovley et al., 1996; Lovley et al., 1998; Lovley et al. 2000).
Recovery of AQDS-reducing microorganisms either through enrichment and isolation
procedures or dilution-to-extinction approaches yield organisms that also can reduce
iron [Fe(III); Coates et al., 1998)]. The reduction of AQDS to AHQDS is visually
apparent as the conversion of the relatively colorless AQDS to the orange, AHQDS.
Fe(III)- and Mn(IV)-reducing microorganisms can be obtained in pure culture
through standard anaerobic approaches of isolating colonies in tubes or on plates or
through dilution-to-extinction in liquid media. Soluble Fe(III) forms or AQDS are
often used for isolating colonies on agar-solidified media, but colonies also can be
obtained by incorporating Fe(III) and Mn(IV) oxides into solidified media. The
Fe(III)- and Mn(IV)-reducing microorganisms that have the ability to use other
electron acceptors often can be successfully purified from Fe(III)- or Mn(IV)-reducing