279. 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
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 (Fe3O4) (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 (FeCO3) and rhodochrosite (MnCO3) 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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 c3, 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 c3 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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 -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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 -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 -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.
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The FMR in the -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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 (Fe3O4) siderite (FeCO3),
vivianite (Fe3PO4· 8H2O) and rhodochrosite (MnCO3; 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
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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).
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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.,
1998b). 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
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 also may 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 [2-14C]-acetate
(Lovley, 1997a). The reduction of Fe(III) can be considered to be the TEAP if: 1) a tracer quantity of [2-14C]-acetate
added to the sediments is converted to 14CO2 with no production of 14CH4; 2) the production of 14CO2 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 14CH4 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
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
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 enrichment cultures with
these alternative electron acceptors. Common alternative electron acceptors include nitrate, fumarate, sulfur, and oxygen.
Suggested Media for Enrichment and Culturing of FMR
Freshwater and marine media suitable for culturing a diversity of mesophilic FMR are described below. A variety of other
media have also been used which can be found in the references for the individual organisms. The media described here
have a bicarbonate-carbon dioxide buffer system and the headspace gas typically contains 20% carbon dioxide to establish
an initial pH of ca. 6.8.
PROCEDURE: Freshwater Medium
To 900 ml water add:
NaHCO3
2.50 g
NH4Cl
0.25 g
NaH2PO4· H2O 0.60 g
KCl
0.10 g
Vitamin Solution 10.0 ml
Mineral Solution 10.0 ml
Bring solution to a final volume of 1 liter. Media is dispensed, sparged with an 80:20 mixture of N2:CO2 gas and then
autoclaved.
PROCEDURE: Marine Medium
Medium contains, per liter:
NaCl
KCl
NaHCO3
20.0g
0.67g
2.5.0 g
Vitamin solution 10.0 ml
Mineral solution 10.0 ml
RST minerals stock 20.0 ml
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
Salt stock*
50.0 ml
*Add salt solution aseptically and anaerobically after autoclaving.
PROCEDURE: RST Minerals Stock 50X
Stock contains, per 100 ml:
NH4Cl
KCl
KH2PO4
5.0 g
0.5 g
0.5 g
MgSO4· 7H2O 1.0 g
CaCl2· 2H2O 0.1 g
PROCEDURE: Salt Stock
Stock contains, per 100 ml:
MgCl2· 6H2O 21.2 g
CaCl2· 2H2O 3.04 g
PROCEDURE: Vitamin Solution
Solution contains, per liter:
Biotin
2.0 mg
Folic acid
2.0 mg
Pyridoxine HCl
10.0 mg
Riboflavin
5.0 mg
Thiamine
5.0 mg
Nicotinic acid
5.0 mg
Pantothenic acid
5.0 mg
B-12
0.1 mg
p-Aminobenzoic acid 5.0 mg
Thioctic acid
5.0 mg
PROCEDURE: Mineral Solution
grams per liter
Trisodium nitrilotriacetic acid 1.5 g
MgSO4
3.0 g
MnSO4· H2O
NaCl
FeSO4· 7H2O
0.5 g
1.0 g
0.1 g
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
CaCl2· 2H2O
0.1 g
CoCl2· 6H2O
0.1 g
ZnCl2
0.13 g
CuSO4· 5H2O
0.01 g
AlK(SO4)2· 12H2O
0.01 g
H3BO3
0.01 g
Na2MoO4
0.025 g
NiCl2· 6H2O
0.024 g
Na2WO4· 2H2O
0.025 g
Preparation of Fe(III) and Mn(IV) Forms
Poorly Crystalline Fe(III) Oxide
Dissolve FeCl3·6H2O in water to provide final concentration of 0.4M. Stir continually while SLOWLY adjusting the pH
to 7.0 dropwise with 10 M NaOH solution. It is extremely important not to let the pH rise above pH 7 even momentarily
during the neutralization step because this will result in an Fe(III) oxide that is much less available for microbial
reduction. Continue to stir for 30 minutes once pH 7 is reached and recheck pH to be sure it has stabilized at pH 7. To
remove dissolved chloride, centrifuge the suspension at 5,000 rpm for 15 minutes. Discard the supernatant, resuspend the
Fe(III) oxide in water, and centrifuge. Repeat six times. On the last wash, resuspend the Fe(III) oxide to a final volume of
approximately 400 ml, and after determining iron content, adjust Fe(III) concentration to approximately 1 mole per liter.
Typically, Fe(III) oxide is added to individual tubes of media to provide 100 mmol per liter.
Fe(III)-Citrate
Prior to the addition of any of the media constituents, heat 800 ml of water on a stirring hot-plate to near boiling. Add
Fe(III)-citrate [typically 13.7 g to provide a final concentration of ca. 50 mM Fe(III)]. Once the ferric citrate is dissolved
quickly cool the medium to room temperature in an ice bath. Adjust pH to 6.0 using 10N NaOH. When the pH approaches
5.0, add the NaOH dropwise. Add medium constituents as outlined above. Bring to a final volume of 1 liter. Do not
expose this media to direct sunlight to prevent photoreduction of the Fe(III).
Fe(III) Nitrilotriacetic Acid
To make a stock of 100 mM Fe(III)-NTA, dissolve 1.64 g of NaHCO3 in 80 ml water. Add 2.56 g C6H6NO6Na3 (sodium
nitrilotriacetic acid) and then 2.7 g FeCl3·6H2O. Bring the solution up to 100 ml. Sparge the solution with N2 gas and
filter sterilize into a sterile, anaerobic serum bottle. Do not autoclave. Typically, 100 mM Fe(III)-NTA stock is added to
individual tubes of media to provide a final concentration of 5 or 10 mmol of Fe(III).
Goethite
Prepare a 0.4M FeCl3·6H2O solution. With continual stirring, adjust the pH to between 11 and 12 with 10 M NaOH
solution. The suspension will become very thick. Ensure continual stirring and rinse the pH electrode frequently. The
color of this suspension will turn to an ochre color as goethite is formed. One week at room temperature followed by 16
hours at 90°C is sufficient to convert the Fe(III) to goethite. The suspension should be washed to remove chloride, as
described above for poorly crystalline Fe(III) oxide. The formation of goethite should be confirmed by X-ray diffraction
analysis. The Fe(III) oxide also should be tested with extractants (Lovley and Phillips, 1987b; Phillips and Lovley, 1987)
to ensure that it does not contain poorly crystalline Fe(III) oxide.
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279. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes
Hematite
Hematite is readily available from chemical supply companies as "Ferric Oxide."
Manganese Oxide
To l liter of a solution containing 80 mM NaOH and 20 mM KMnO4 slowly add l liter of 30 mM MnCl2 with mixing.
Wash the manganese oxide precipitate, as described above for poorly crystalline Fe(III) oxide, to lower the dissolved
chloride concentration.
Enumeration of Fe(III)- and Mn(IV)-reducing Microorganisms
The FMR in environments can be enumerated with standard most-probable-number (MPN) culturing techniques using
variations of media described above. Enumerations typically use Fe(III) or AQDS as the electron acceptor with the
understanding that the Fe(III)-reducing microorganisms recovered are likely to have the ability to reduce Mn(IV) as well.
Poorly crystalline Fe(III) oxide or Fe(III)-NTA is preferred over Fe(III)-citrate and Fe(III)-pyrophosphate, which promote
the growth of fermentative microorganisms. One successful approach has been to add a combination of poorly crystalline
Fe(III) oxide (100 mmol/liter) and 4 mM NTA to provide a supply of chelated Fe(III).
FMR also can be counted in plate counts in which Fe(III)-NTA or AQDS has been added as the electron acceptor.
Clearing zones develop around FMR reducing Fe(III)-NTA, and growth with AQDS as the electron acceptor results in the
formation or orange colonies or zones.
When possible, molecular enumeration rather than viable culturing enumeration techniques are the preferred methods
because of the potential biases associated with the latter. The wide phylogenetic diversity of dissimilatory Fe(III) reducing
microorganisms and the lack of an identified conserved gene associated with Fe(III) reduction make it impossible to
enumerate Fe(III)-reducing microorganisms with one specific gene sequence (Lonergan et al., 1996). However, target 16S
rRNA sequences that are selective for known groups of Fe(III)-reducing microorganisms have been identified and have
been used to study the distribution of Fe(III)-reducing microorganisms in sedimentary environments (DiChristina and
DeLong, 1993; Anderson et al., 1998; Rooney-Varga et al., 1999; Synoeyenbos-West et al., 1999).
SUMMARY
Microbial reduction of Fe(III) and Mn(IV) is of environmental significance in a variety of aquatic sediments and the
subsurface, influencing both the carbon cycle and the fate of many metals and metalloids, in both pristine and
contaminated environments. Geological and microbiological evidence suggests that Fe(III) reduction was one of the
earliest forms of respiration. A wide phylogenetic diversity of Fe(III)- and Mn(IV)-reducing microorganisms have been
recovered in pure culture, but with the exception of the recently recognized importance of Geobacter in subsurface
environments, little is known about the distribution or relative contributions of the various Fe(III)-reducing
microorganisms. The study of the mechanisms of Fe(III) and Mn(IV) reduction are also in their infancy. However, now
that methods for culturing these organisms are well-developed, it seems likely that increased insight into the
ecophysiology of Fe(III)- and Mn(IV)-reducing microorganisms is forthcoming.
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