World Journal of Microbiology & Biotechnology 19: 215–225, 2003.
Ó 2003 Kluwer Academic Publishers. Printed in the Netherlands.
215
Review: microbial mechanisms of accessing insoluble Fe(III) as an energy source
Y-Su Luu and Juliana A. Ramsay*
Department of Chemical Engineering, Queen’s University, Dupuis Hall, Division Street, Kingston, Ont., Canada K7L
3N6
*Author for correspondence: Tel.: +1-613-533-2770, Fax: +1-613-533-6637, E-mail: [email protected]
Received 9 October 2002; accepted 2 December 2002
Keywords: Cell/mineral contact, dissimilatory iron-reducing bacteria, electron shuttling, Fe(III) reduction, humic
substances
Summary
Of all the terminal electron acceptors, Fe(III) is the most naturally abundant in many subsurface environments.
Fe(III)-reducing microorganisms are phylogenetically diverse and have been isolated from a variety of sources.
Unlike most electron acceptors, Fe(III) has a very low solubility and is usually present as insoluble oxides at neutral
pH. The mechanisms by which microorganisms access and reduce insoluble Fe(III) are poorly understood. Initially,
it was considered that microorganisms could only reduce insoluble Fe(III) through direct contact with the oxide.
However, recent studies indicate that extracellular electron shuttling or Fe(III)-chelating compounds may alleviate
the need for cell–oxide contact. These include microbially secreted compounds or exogenous electron shuttling
agents, mainly from humic substances. Electron shuttling via humic substances is likely a significant process for
Fe(III) reduction in subsurface environments. This paper reviews the various mechanisms by which Fe(III)
reduction may be occurring in pure culture and in soils and sediments.
Introduction
Until recently, it was believed that Fe(III) reduction in
sedimentary and subsurface environments was the result
of abiotic processes, whereby changes in pH and/or
redox potential promoted the conversion of Fe(III) to
Fe(II) (Fenchel & Blackburn 1979). It is now known
that microbial respiration with Fe(III) oxides as the
terminal electron acceptor is an important process in
anoxic environments, and that nearly all the Fe(III)
reduction in these environments is enzymatically catalyzed (Lovley 1993; Lovley 1997). Fe(III) respiration is
commonly referred to as dissimilatory iron reduction
indicating that Fe(III) reduction is not necessarily linked
to energy conservation. However, if energy is conserved,
it should be referred to as Fe(III) respiration. In all
cases, Fe(III) is reduced to Fe(II).
Microbial reduction of Fe(III) oxides has several
significant environmental impacts. Water quality and
soil chemistry can be affected (Lovley 2000). For
example, the increase of dissolved Fe(II) in groundwater
as a consequence of microbial Fe(III) reduction affects
the taste of drinking water, gives rise to plumbing and
staining problems and can be expensive to remediate.
The release of a wide variety of trace toxic metals which
adsorb onto, or co-precipitate with, Fe(III) oxides is a
potential hazard when microbial Fe(III) reduction
occurs (Lovely 1991). The degradation of naturally
occurring organic compounds and organic contaminants by Fe(III)-reducing microbes has also been
documented.
Fe(III) is the most abundant of all the available
terminal electron acceptors in many subsurface environments, consisting of several percent of the total
sediment by weight (Lovley 1991). From a thermodynamic point of view, Fe(III) reduction is energetically
favorable compared with sulfate reduction and methanogenesis, but yields less free energy than O2 and nitrate
reduction (Table 1). In most anaerobic environments,
Fe(III) reduction occurs before sulfate reduction and
methanogenesis but is inhibited in the presence of nitrate
(Tugel et al. 1986; Lovley & Phillips 1987; Lovley 1991).
At neutral pH, the majority of Fe(III) occurs as
insoluble oxides. This review focuses on the mechanisms
of microbial electron transport to the insoluble Fe(III)
oxides by Fe(III)-reducing microbes.
Fe(III) reduction
Dissimilatory Fe(III)-reducing bacteria
The first dissimilatory Fe(III)-reducing bacteria discovered were fermentative, and did not require Fe(III) for
growth. They were capable of reducing Fe(III) while
fermenting sugars or amino acids and generating
216
Y-Su Luu and J.A. Ramsay
Table 1. Comparison of free energy released from the oxidation of
organic carbona using different terminal electron acceptors (adapted
from Froelich et al. 1979).
Electron acceptor
Free energy (kJ/mol glucose)
O2
Nitrate
Fe(III) (hematite)
Fe(III) (goethite)
Sulfate
CO2
)3190
)3030
)1410
)1330
)380
)350
a
Organic carbon of the formula (CH2O)106(NH3)16(H3PO4).
acetate, alcohols, H2 and other fermentation products.
However, less than 5% of the reducing equivalents from
the metabolized substrates was transferred to Fe(III)
(Lovley 1991).
Only a small percentage of Fe(III) reduction in
sedimentary environments can be attributed to fermentative microorganisms. The majority of Fe(III)
reduction is due to the oxidation of fermentation endproducts such as acetate (Lovley 1993). Geobacter
metallireducens was the first pure culture shown to
completely oxidize acetate with Fe(III) as the electron
acceptor (Lovley et al. 1993). This organism, a member
of the d subclass of Proteobacteria, is a strict anaerobe
isolated from freshwater sediments, capable of oxidizing
several organic acids and alcohols using Fe(III) as the
electron acceptor. Other members of this genus have
been identified, all capable of conserving energy through
Fe(III) reduction (Lovley 2000; Coates et al. 2001).
Members of the genera Desulfuromonas (Roden &
Lovley 1993; Coates et al. 1995), Pelobacter (Lovley
et al. 1995) and Desulfuromusa (Fredrickson & Gorby
1996) also reduce Fe(III). As with Geobacter, these
genera all belong to the d subdivision of Proteobacteria,
suggesting that members of the Geobacteraceae family
share a common Fe(III)-reducing ancestor (Lonergan
et al. 1996).
Although most of the known Fe(III)-reducing microorganisms belong to the d subdivision of Proteobacteria,
the ability to reduce Fe(III) is not limited to Geobacteraceae. In the c subclass of Proteobacteria, organisms
such as Shewanella putrefaciens, Shewanella alga, and
Ferrimonas balearica can generate energy for growth
from the reduction of Fe(III) with H2 or organic acids
serving as the electron donor (Lovley 2000). Fe(III)
reducers also occur outside of the Proteobacteria.
Geovibrio ferrireducens (Caccavo et al. 1996) and Geothrix fermentans (Coates et al. 1999) are two phylogenetically distinct Fe(III)-reducing bacteria. Geothrix
ferrireducens was isolated from a hydrocarbon-contaminated soil while Geothrix fermentans was isolated from
the Fe(III)-reducing zone of shallow contaminated
aquifers. Geovibrio ferrireducens and Geothrix fermentans are not closely related to each other or to any other
previously described bacteria. The phylogenetic placement of these novel organisms suggests that the ability
to reduce Fe(III) is spread throughout the domain
Bacteria (Lonergan et al. 1996). The capacity for Fe(III)
reduction is also present in all hyperthermophiles
evaluated thus far, both in the Bacteria and the Archaea
(Lovley 2000; Lovley et al. 2000; Childers & Lovley
2001; Zhou et al. 2001; Kashefi et al. 2002). Although
knowledge of the diversity of Fe(III)-reducing organisms continues to expand, relatively little is known about
which of these organisms are most important in different
sedimentary environments.
Fe(III) cycling
Even in anaerobic environments where Fe(III) is not
abundant, Fe(III) reduction may still be the dominant
electron accepting process due to iron cycling processes.
Iron is a unique terminal electron acceptor in that it can
be readily reoxidized and returned to sediments via
settling. This provides a mechanism for Fe(III) regeneration and for the movement of oxidizing potential into
anaerobic zones. In the coastal sediments near Denmark, Fe(III) recycling was rapid and each molecule of
Fe(III) was estimated to be recycled 100–300 times
before being buried in sediments (Canfield et al. 1993).
Therefore, using Fe(III) concentrations to predict the
importance of Fe(III) respiration in anaerobic environments may be misleading.
Some of the different reactions that iron undergoes in
nature are depicted in Figure 1. Fe(III) oxides, Fe(III)
oxides complexed with trace metals and ferric phosphate
settle to the anoxic zone where Fe(III) reduction may be
coupled to organic carbon oxidation, generating soluble
Fe(II) and releasing any bound phosphate and metals.
Some of the Fe(II) may form insoluble precipitates such
as siderite (FeCO3), pyrite (FeS2), vivianite [Fe3(PO4)2]
and magnetite (Fe3O4) while some may diffuse into the
Figure 1. Iron cycling in aquatic environments. Step 1: Solid oxides
settle in suboxic zone. Step 2: Phosphate and metals are removed via
precipitation and complexation. Step 3: Organic carbon oxidation
using iron as terminal electron acceptor. Step 4: Organic phosphate
and metals released during iron reduction. Step 5: Some Fe(II) forms
insoluble precipitates in suboxic zone. Step 6: Some Fe(II) diffuses into
oxic zone. Step 7: Reoxidation of Fe(II) occurs to form insoluble
oxides. Step 8: If there is no input of organic carbon, oxides
accumulate in sediments of suboxic zone, otherwise the cycle continues
at step 1 (adapted from Nealson & Saffarini 1994). With permission
from Annual Review of Microbiology, Volume 48 Ó 1994 by Annual
Reviews www.annualreviews.org
217
Accessing insoluble Fe(III) as an energy source
overlying oxic zone where they are reoxidized to form
insoluble Fe(III) oxides to possibly continue the cycle.
This regeneration and distribution of Fe(III) is driven
largely by the input of organic matter. When no organic
matter is present, the Fe(III) oxides accumulate in the
sediment with little iron cycling.
Because the majority of microbially reduced Fe(II)
ends up as Fe(II) precipitates in the sediment (Lovley
1991), processes that expose these sediments to O2
stimulate iron reduction by regenerating Fe(III). These
processes include fluctuating water levels in shallow
aquifers, bioturbation by benthic organisms, physical
mixing, reoxidation of Fe(II) in the rhizosphere of
rooted aquatic plants, and the draining of submerged
soils as part of the management of rice paddies (Lovley
2000).
Fe(III) oxides
One important difference between Fe(III) and other
electron acceptors is that it can form different oxides
and hydroxides, each with different crystalline structures
and oxidation states of the metal. Even though an iron
oxide may have the same chemical formula, its crystalline structure, and hence kinetic and thermodynamic
properties, may be different (Nealson & Saffarini 1994).
The form of the Fe(III) has an important effect on its
bioavailability. In general, the more crystalline the
Fe(III) oxide, the less susceptible it is to microbial
reduction.
Several studies suggest that the surface area of the
Fe(III) oxide controls its bioavailability and hence its
rate of reduction. Roden and Zachara (1996) showed
that Shewanella alga BrY reduced amorphous Fe(III)
about 20 times more rapidly than goethite (FeOOH)
which was reduced about 50 times more rapidly than
hematite (Fe2O3). This trend was consistent with surface
area, as the surface area of the amorphous Fe(III) was
greater than that of the goethite, which was greater than
that of the hematite. The authors hypothesized that
Fe(III) reduction depended on cell/mineral contact, and
hence the rate of Fe(III) reduction depended on the
surface area available for microbial attachment. Similar
results were obtained with Shewanella putrefaciens CN32
(Zachara et al. 1998). However, the relationship between
surface area and the rate of Fe(III) reduction is not
simple. Arnold et al. (1986) found that the variation in
the rate of reduction between two forms of hematite was
two- to threefold less than that predicted based on their
surface areas. This indicated that factors such as crystal
structure, morphology, particle aggregation and free
energy may also have an important influence on rates of
Fe(III) reduction, and that surface area should only be
used as a rough indicator of Fe(III) reducibility. Since
both the number of surface sites available for enzymatic
contact and the crystallinity of the oxide correlated
positively with surface area, the relative roles of these
two factors in controlling Fe(III) reduction rates could
not be distinguished (Roden & Zachara 1996).
Although laboratory studies with pure cultures indicate that crystalline Fe(III) oxides, such as goethite and
hematite, are susceptible to microbial reduction, these
forms of Fe(III) may not be reduced in nature. In
sedimentary environments where only crystalline Fe(III)
forms are present, Fe(III)-reducing microorganisms
cannot out-compete sulfate-reducing or methanogenic
microorganisms for electron donors, due to the slow
rates of crystalline Fe(III) reduction (Lovley & Phillips
1987). Studies of Fe(III) reduction in aquatic sediments
showed that the most active zone of Fe(III) reduction
was at the sediment-water interface (up to 4 cm depth),
where Fe(III) was present as amorphous Fe(III) or
poorly ordered Fe(III) oxyhydroxide (Lovley & Phillips
1986). No Fe(III) beyond a 4 cm depth was reduced,
despite the presence of excess electron donors, because
they were highly crystalline Fe(III) oxides (Phillips et al.
1993). Hence, although microorganisms are physiologically able to reduce highly crystalline oxides, it appears
that poorly crystalline Fe(III) oxides, typically present
as coatings on clays or other surfaces, are the primary
source of Fe(III) in the Fe(III)-reducing zone of
subsurface environments.
Mechanisms of enzymatic Fe(III) reduction
Although iron is abundant in many sedimentary environments, Fe(III) has a solubility of approximately
10)9 M at neutral pH, based on recent calculations by
Chipperfield & Ratledge (2000). Hence the majority of
Fe(III) in aquatic sediments is present as insoluble
Fe(III) oxides. The mechanisms of microbial electron
transport to insoluble acceptors are poorly understood.
To respire using Fe(III), the microorganisms must be
able to either (i) attach to the iron substrate and directly
transfer electrons to it, or (ii) solubilize the iron and
deliver it to the electron transport chain, or (iii) use
available electron shuttling compounds or produce their
own. Given the phylogenetic diversity of Fe(III)-reducing microorganisms, it is likely that more than one
strategy for Fe(III) reduction exists.
Requirement for direct cell-mineral contact
If Fe(III)-reducing bacteria are able to secrete extracellular electron shuttles or Fe(III) chelators, then iron
reduction should be possible without the need for direct
cell-mineral contact. A common technique to determine
whether bacterial contact with insoluble Fe(III) is
necessary for reduction to occur has been to separate
the oxides from the microorganisms with dialysis
membranes. The molecular weight cut-offs of these
membranes are chosen such that any soluble chelators
or electron shuttles can pass through but the cells
cannot. When pure cultures of Shewanella putrefaciens
(Arnold et al. 1988), Shewanella alga BrY (Urrutia et al.
1999), Clostridium pasteurianum (Lovley et al. 1991) and
Geobacter metallireducens (Lovley & Phillips 1988;
218
Nevin & Lovley 2000a), and acetate-enrichment cultures
(Tugel et al. 1986) were separated from the insoluble
Fe(III) oxide by dialysis membranes, Fe(III) reduction
did not occur. However, Fe(III) reduction did occur
when there was no separation. This suggests that
microbially secreted, extracellular compounds were not
involved and that direct cell–oxide contact was necessary for Fe(III) reduction to occur. However, when
dialysis membrane experiments were performed with
anthraquinone-2,6-disulfonate (AQDS), an electronshuttling compound, as a positive control, Fe(III)
reduction was not observed (Nevin & Lovley 2000a).
Furthermore, when the Fe(III) oxide was entrapped in
alginate beads with AQDS present, Fe(III) reduction did
occur. This indicates that Fe(III) oxides entrapped in
alginate beads may be a more valid technique to test for
electron-shuttling compounds.
Das and Caccavo (2000) examined the relationship
between cell–oxide adhesion and the rate of Fe(III)
reduction in Shewanella alga BrY. Since both Shewanella alga cells and Fe(III) oxides are negatively charged,
adhesion of cells to the oxides is influenced by electrostatic repulsion. The authors added various concentrations of KCl to balance the repulsive forces to allow the
cells to adsorb to the oxides more readily. The ability of
the cells to reduce insoluble Fe(III) was correlated with
KCl concentration and with the percentage of adhered
cells. These results provide direct evidence that adhesion
is required for Fe(III) reduction by Shewanella alga.
Several lines of circumstantial evidence also suggest
that cells must be in direct contact with the Fe(III)
oxides in order to reduce them. Cell-free filtrates taken
from growing cultures of Shewanella putrefaciens (Arnold et al. 1988), Geobacter metallireducens (Lovley &
Phillips 1988) and Shewanella alga BrY (Caccavo et al.
1992) showed no Fe(III)-reducing activity, suggesting
that iron reduction did not involve an extracellular,
reducing intermediate. A different study with an acetateenrichment culture showed that iron particles were
covered with cells after 72 h of incubation (Tugel et al.
1986). After 7 days, any cells not attached to the Fe(III)
were swollen. Fe(II) accumulation only occurred after
the cells were observed to attach to the Fe(III).
For a variety of Fe(III) oxides, the initial rate and
long-term extent of iron reduction by Shewanella alga
strain BrY (Roden & Zachara 1996) and Shewanella
putrefaciens (Arnold et al. 1986) correlated linearly with
the oxide surface area. Both studies suggest that
bacterial attachment was necessary for Fe(III) reduction
to occur, and that the oxide surface area correlated
directly with the concentration of surface sites available
for enzymatic contact. Consistent with this hypothesis is
the finding that Fe(II) adsorption to the iron oxide
lowered the extent of Fe(III) reduction, presumably
blocking the surface sites for bacterial attachment. In
semi-continuous culture, the elimination of accumulated
Fe(II) by Shewanella alga BrY by convection significantly improved the rate and extent of Fe(III) reduction
compared with batch cultures (Roden & Urrutia 1999).
Y-Su Luu and J.A. Ramsay
Similarly, the addition of Fe(II)-sorbing minerals, such
as aluminum oxides and layered silicates, improved
Fe(III) reduction probably by acting as Fe(II) sinks
preventing the Fe(II) from sorbing onto the oxide
surface (Urrutia et al. 1999).
Fe(II) biosorption by dissimilatory iron-reducing
bacteria may also affect the rate and extent of Fe(III)
reduction. Shewanella alga BrY cells precoated with
Fe(II) reduced Fe(III) more slowly than untreated cells
(Urrutia et al. 1998). The rate of Fe(III) reduction by
Fe(II)-coated cells was equivalent to that of cells from
cultures that were in the later stages of growth. These
results suggest that the sorption of Fe(II) and the
precipitation of Fe(II)/Fe(III) solids on the surface of
the cell interferes with electron transport to Fe(III)
oxides. The Fe(II) sorption capacity of the cells was
greatest when they were in direct contact with the
oxides. Under non-growth conditions, the Fe(II) sorption capacity of the cells was less when Shewanella alga
and Fe(III) oxides were in separate dialysis membranes
than when the cells and oxides were allowed to interact
directly. This enhanced ability of the cells to sorb Fe(II)
when they are in contact with the oxides may be due to
the production of exopolysaccharide (EPS) which has a
high affinity for binding metal cations. The production
of EPS was stimulated when the cells were in the
presence of Fe(III). A separate study with Shewanella
putrefaciens CN32 growing on Fe(III)-citrate with
aqueous Fe(II) initially present had a lag phase before
the onset of Fe(III) reduction (Liu et al. 2001). During
the lag phase, iron mineral precipitation was observed
on the cells. However, once in active growth, all cells
were free of surface precipitates. This recovery from
Fe(II) inhibition may be due to the cells’ capacity to
shed the sorbed Fe(II), perhaps through the formation
of membrane vesicles.
While strong evidence supports the view that direct
cell–oxide contact is a prerequisite for Fe(III) reduction
for many species, the relationship between the Fe(III)reduction rate and the amount of cell–oxide contact is
not a straightforward one. Caccavo et al. (1997) compared the rate of iron reduction between an adhesiondeficient strain of Shewanella alga and the wild-type
Shewanella alga BrY. Although less than 50% of the
mutant cells adhered, Fe(III) was reduced at an identical
rate to that of the wild type with 100% adherent cells.
These results indicate that bacterial adhesion to iron
oxides may not be necessary for Fe(III) reduction to
occur. The authors suggest that Shewanella alga may
produce a surface protein that can efficiently chelate
Fe(III). However, such a mechanism has yet to be
proven.
Membrane-bound electron transport chains
Functions, such
associated with
Soluble electron
the cell through
as respiratory electron transport, are
the bacterial cytoplasmic membrane.
acceptors, such as O2 or nitrate, enter
passive diffusion. However, at neutral
219
Accessing insoluble Fe(III) as an energy source
pH, Fe(III) is insoluble and cannot diffuse into the cell.
For Gram-negative bacteria to reduce Fe(III) oxides,
they must somehow establish a link between the
insoluble oxides and the electron transport system in
the cytoplasmic membrane. Shewanella putrefaciens
MR-1 may achieve this by localizing c-type cytochromes
in its outer membrane where they could potentially
make contact with Fe(III) oxides (Myers & Myers 1992,
1997; Beliaev et al. 2001). When Shewanella putrefaciens
was grown under aerobic conditions, the cytochrome
content was highest in the cytoplasmic membrane. In
contrast, when grown anaerobically, approximately
80% of the membrane-bound cytochromes were localized in the outer membrane (Myers & Myers 1992).
Anaerobically grown cells also had significant amounts
of periplasmic cytochromes, which could link the
electron transport components of the cytoplasmic membrane and the outer membrane. In a different study,
ferric reductase activity for Shewanella putrefaciens MR1 was detected exclusively in the membrane fractions of
anaerobically grown cells, with about 55% in the outer
membrane. No ferric reductase activity was detected in
aerobically grown cells (Myers & Myers 1993). Recent
work suggests that a 91 kDa heme-containing protein
associated with the outer membrane fraction of Shewanella putrefaciens strain 200 is involved in Fe(III)
reduction (DiChristina et al. 2002). The gene encoding
secretion of this protein into the outer membrane has
been identified, providing the first genetic evidence
linking outer membrane-targeted proteins to dissimilatory Fe(III) reduction (DiChristina et al. 2002).
Gaspard et al. (1998) also found ferric reductase
activity in the membrane fraction of Geobacter sulfurreducens with about 80% located in the outer membrane. Evidence suggests that this reductase complex
contained a c-type cytochrome and required NADH as
an electron donor (Magnuson et al. 2000, 2001). This
cytochrome was able to undergo reoxidation upon
addition of Fe(III) oxyhydroxide, indicating that it can
pass electrons to Fe(III). A preliminary model for
Figure 2. Proposed model for electron transport to extracellular
Fe(III) in G. sulfurreducens (Lovley 2000 with permission from ASM
Press, Washington DC).
electron transport to insoluble Fe(III) in Geobacter
species has been proposed (Figure 2) (Lovley 2000). In
this model, a NADH hydrogenase localized in the inner
membrane is part of a respiratory complex that contains
an 89 kDa c-type cytochrome. This cytochrome can
transfer electrons to a 9 kDa periplasmic cytochrome,
which can in turn transfer electrons to a 41 kDa
membrane-bound cytochrome. Because the 41 kDa
cytochrome is associated with the outer membrane, it
is able to contact insoluble Fe(III) and donate electrons
to Fe(III).
Finding and accessing insoluble, poorly diffusible
Fe(III) in order to reduce it poses a challenge for cells
with limited mobility. Geobacter metallireducens appears
to overcome this obstacle by synthesizing lateral flagella
and pili when grown with insoluble Fe(III) as the
terminal electron acceptor. Neither flagella nor pili was
observed when soluble Fe(III) was used (Childers et al.
2002). Thus, flagellar synthesis may be an effective
strategy for accessing insoluble Fe(III) for dissimilatory
Fe(III)-reducing bacteria that cannot excrete electron
shuttling compounds or Fe(III) chelators. In many
environments, synthesis of electron shuttles may not be
energetically feasible if they are lost upon release by
diffusion or advection. Mobile dissimilatory Fe(III)reducing bacteria would have the competitive advantage
in such situations. This may explain the predominance
of Geobacter species over Shewanella and Geothrix
species in many subsurface environments (Childers et al.
2002).
Siderophores
Most microorganisms require iron for essential processes such as the synthesis of ribonucleotide sequences and
the formation of a range of heme- and non-hemeproteins. Since Fe(III) is insoluble in most natural
environments, many microbes rely on high-affinity
Fe(III) chelators, siderophores, for Fe(III) assimilation.
Siderophores are likely to be produced by all aerobic
and facultative microorganisms, but not by strict
anaerobes or lactic acid bacteria (Crichton & Charloteaux-Wauters 1987). They are water-soluble, low molecular weight (500–1000 Da), Fe(III)-specific ligands
which are induced at low iron concentrations (Guerinot
1994; Ratledge & Dover 2000). They facilitate Fe(III)
solubilization and transport into the cell.
Although there is a wide structural variation among
siderophores, they may generally be classified as
hydroxamates or phenolates/catecholates (Crichton &
Charloteaux-Wauters 1987). The iron is usually bound
to the siderophore by the O2 atoms of these functional
groups. The formation constants for typical molecules
containing three bidentate ligands is in the order of
1030–1050 (Neilands 1995). The assimilation of Fe(III)
consists of three stages, beginning with the secretion of
siderophores in response to low concentrations of iron.
Once secreted, the siderophore chelates the Fe(III)
and the iron–siderophore complex binds to specific
220
membrane receptors. Finally, the iron is released from
the siderophore to the cell.
There are three proposed schemes for the removal of
iron from the siderophore (Hughes & Poole 1989;
Ratledge & Dover 2000). In the first mechanism, the
iron–siderophore complex is transported across the cell
membrane. The iron is released intracellularly via a
reduction step as the siderophore has a low affinity for
Fe(II). This leaves an intact ligand which can again be
excreted and reused for iron transport. In the second
mechanism, the iron–siderophore complex is taken up
into the cell, and the release of the metal is accomplished
through chemical modification or destruction of the
ligand. In the third mechanism, the siderophore acts as
an iron shuttle bringing the iron to the cell membrane
but does not enter the cell. Instead, the iron is donated
to a second membrane-bound chelator followed by
reduction, and the siderophore remains extracellular.
It is generally assumed that the enzymes used for
dissimilatory Fe(III) reduction are distinct from the
enzymes involved in iron–siderophore transport for
Fe(III) assimilation. However, not enough is known
about either type of enzymes to rule out possible
similarities. The effectiveness of siderophores in making
insoluble Fe(III) bioavailable for assimilation may also
be important in making Fe(III) available as a terminal
electron acceptor during respiration. Recent studies with
Shewanella alga BrY (Nevin & Lovley 2002a) and
Geothrix fermentans (Nevin & Lovley 2002b) found that
these cultures solubilized significant quantities of Fe(III)
during the most active phase of Fe(III) reduction. These
results suggest that the cells secreted Fe(III)-solubilizing
compounds, possibly siderophores, as a mechanism to
access and reduce insoluble Fe(III). However, the high
affinity of siderophores for Fe(III) may pose a challenge
in Fe(III) respiration. When Geobacter metallireducens
was grown on insoluble Fe(III), the addition of hydroxamate siderophores did not stimulate cell growth (Holmen
et al. 1999). This may be due to the high strength and
redox stability of the Fe(III)–hydroxamate complex,
impeding reduction by Geobacter metallireducens.
Microbially secreted electron shuttling compounds
Recent evidence indicates that Fe(III) reduction may
involve extracellular cytochromes which shuttle electrons to insoluble Fe(III). Geobacter sulfurreducens
produces a periplasmic c-type cytochrome which was
found in equal amounts in the membrane fraction, the
periplasmic space and, extracellularly, in the growth
medium (Seeliger et al. 1998). This cytochrome reduced
various soluble and insoluble Fe(III) at high rates and
also transferred electrons to partner bacteria. A coculture was established whereby the c-type cytochrome
reduced by Geobacter sulfurreducens was re-oxidized by
Wolinella succinogenes in the presence of nitrate. However, this role for an extracellular cytochrome has been
questioned, partly because of the high energetic cost of
such a mechanism (Lloyd et al. 1999).
Y-Su Luu and J.A. Ramsay
Shewanella oneidensis MR-1 (formerly Shewanella
putrefaciens MR-1) secretes a small, non-proteinaceous
compound which has the characteristics of a quinone
and functions as an electron shuttle, thereby enabling
S. oneidensis to respire using oxidized humic acids
(Newman & Kolter 2000). Such a compound may also
be involved in extracellular electron transfer to Fe(III)
oxides. However, respiration using insoluble Fe(III)
minerals by this route has yet to be proven.
Nevin and Lovley (2002b) investigated the mechanisms of Fe(III) reduction by Geothrix fermentans, an
organism isolated from the Fe(III)-reducing zone of
petroleum-contaminated aquifers and phylogenetically
distinct from Geobacteraceae (Anderson et al. 1998).
Unlike Geobacter metallireducens, Geothrix fermentans
was able to reduce Fe(III) oxides when the Fe(III) was
incorporated into microporous alginate beads, which
prevented direct contact between cells and oxides. After
35 days, approximately 25 mM Fe(II) was produced
when the Fe(III) oxide was entrapped in alginate beads,
compared with 35 mM with free Fe(III) oxide. Addition
of culture filtrates to washed cell suspensions of Geothrix fermentans also stimulated Fe(III) reduction. These
results suggest that Geothrix fermentans can secrete an
extracellular compound that can shuttle electrons between the cells and the poorly crystalline Fe(III) oxides.
Thin-layer chromatography indicated that this compound has characteristics similar to a water-soluble
quinone.
Further evidence for the production of electron
shuttles by dissimilatory Fe(III)-reducing bacteria
comes from studies on Shewanella alga. Turick et al.
(2002) reported that Shewanella alga BrY produced an
extracellular melanin that contains quinoid compounds
that could act as the sole terminal electron acceptor for
growth. Bacterially reduced melanin can also reduce
Fe(III) oxides in the absence of cells suggesting that the
melanin can act as an electron shuttle between the cells
and Fe(III) oxides. Only a small amount of melanin was
required to significantly enhance the rate of Fe(III)
reduction. The ability to produce melanin may provide
Shewanella alga with an effective strategy for reducing
insoluble Fe(III) oxides and for growing in anaerobic
environments which lack other terminal electron acceptors. In a separate study, Nevin and Lovley (2002a)
showed that Shewanella alga BrY reduced amorphous
Fe(III) oxide entrapped within alginate beads, providing
additional evidence that Shewanella alga can produce
extracellular electron-shuttling compounds.
Fe(III) reduction in sedimentary environments
Although pure cultures of dissimilatory Fe(III)-reducing
bacteria can reduce Fe(III) by direct enzymatic processes, the pathways of Fe(III) reduction in natural environments are more complex. Microbially reduced
U(VI), S0 and extracellular quinones can act as electron
shuttles to Fe(III), suggesting that much of the Fe(III)
221
Accessing insoluble Fe(III) as an energy source
reduction in sedimentary environments may occur
non-enzymatically. Of all these potential shuttling compounds, only extracellular quinones significantly stimulate Fe(III) reduction in aquifer sediments (Nevin &
Lovley 2000b). The most common source of extracellular quinones in nature is from humic substances.
Humic substances
Humic substances are ubiquitous in soils and sediments
and are formed from the degradation of plants, animals
and microorganisms. They are a heterogeneous mixture
of many compounds with no defined chemical structure.
Their physical and chemical characteristics depend on
the age and source of the material. Humic substances
can be described as dark-coloured, acidic, predominantly aromatic, hydrophilic, chemically complex, polyelectrolyte-like materials that range in molecular weight
from a few hundred to several thousand Daltons
(Schnitzer 1978). The major oxygen-containing functional groups in humic substances are carboxylic acids,
phenolic and alcoholic hydroxyls, ketones and quinones
(Schnitzer 1978). Humic substances are considered to be
inert, refractory organic material, particularly under
anaerobic conditions.
Based on solubility characteristics, humic substances
can be divided into three major fractions: humin, humic
acid and fulvic acid (Aiken et al. 1985). Humin is the
water-insoluble fraction at any pH value; humic acid is
water-insoluble under highly acidic conditions (pH
below 2); and fulvic acids are soluble at all pHs. The
majority of the literature on humic substances focuses on
humic and fulvic acids, with relatively little about humin.
Compared to humic acids, fulvic acids have lower
molecular weights, and a higher O but lower C content.
Fulvic acids also contain more acidic functional groups,
particularly COOH (Stevenson 1985). Humin, which
cannot be extracted into solution, is believed to exist as
high-molecular-weight polymers or is closely associated
with silicates and other minerals (Stevenson 1982).
Humic substances are of interest from an environmental perspective because of their interactions with
contaminants in aquatic environments. The binding of
non-polar, hydrophobic compounds by humic substances increases their solubility and thus their mobility
(Caron & Suffet 1989). The persistence, leaching and
bioavailability of hydrophobic contaminants, such as
pesticides, are affected by their adsorption onto soil
organic matter (Khan 1978). Humic substances also
influence the toxicity and transport of heavy metals.
They can form complexes with metal ions and also alter
the speciation of the metal through oxidation–reduction
reactions (Weber 1988; Manahan 1989). A new role for
humic acids as electron acceptors for microbial respiration has been reported (Lovley et al. 1996a). This form
of respiration has received much attention because of its
significance in the fate of organic pollutants in the
environment.
Humic substances as electron acceptors
Humic substances are the most abundant form of
organic matter in many sedimentary environments.
They can serve as electron acceptors for many bacteria
and may be an important form of respiration under
anaerobic conditions (Cervantes et al. 2000a). Humicreducing bacteria have been recovered from diverse
environments (Coates et al. 1998) and include hyperthermophilic (Lovley et al. 2000) and fermenting bacteria (Benz et al. 1998). Thus far, all microorganisms that
can use humics as electron acceptors can also reduce
Fe(III) (Lovley 2000).
The presence of humic substances significantly improves Fe(III) reduction when coupled to organic
carbon oxidation (Lovley et al. 1996a, b; Anderson &
Lovley 1999). This stimulation was thought to be due to
chelation of Fe(III) by humics (Lovley et al. 1996b) but
subsequent studies indicate that too little Fe(III) was
solubilized to account for the large improvements in
Fe(III) reduction observed (Lovley et al. 1996a; Lovley
& Blunt-Harris 1999). Lovley et al. (1996b) demonstrated that humic substances stimulated Fe(III) reduction
by serving as electron shuttles between the cells and
Fe(III) oxides. In this model, dissimilatory iron-reducing bacteria oxidize organic compounds by transferring
electrons to humic substances. The reduced humics can
then abiotically transfer electrons to Fe(III), regenerating the oxidized form of the humics (Figure 3). This
electron shuttling alleviates the need for the bacteria to
establish direct contact with the Fe(III) oxides to reduce
them.
Quinone moieties appear to be the important electronaccepting group for microbial respiration on humic
substances. Electron spin resonance measurements show
a direct correlation between the electron-accepting
capacity of humics and the number of quinone groups
(Scott et al. 1998). Microorganisms which have been
shown to reduce humic substances can also reduce the
model compound, AQDS, a humic acid analogue,
providing further evidence that extracellular quinones
can serve as electron acceptors for microbial respiration
(Coates et al. 1998; Lovley et al. 1998; Cervantes et al.
2000b). Studies with Geobacter metallireducens and
Shewanella alga indicate that the cells transfer two
electrons to AQDS, generating anthrahydroquinone2,6-disulphonate (AHQDS). AHQDS can then shuttle
Figure 3. Model for humic acid stimulation of Fe(III) reduction
(Lovley et al. 1996a, by permission from Nature, Volume 382 Ó 1996,
Macmillan Publishers Ltd., www.nature.com/nature).
222
electrons to Fe(III), re-generating AQDS (Lovley et al.
1996a).
Evidence suggests that electron shuttling by humic
substances is an important mechanism for Fe(III)
reduction in subsurface sediments (Nevin & Lovley
2000b). The addition of soil humic acids or AQDS to
cultures of Geobacter metallireducens resulted in significant reduction of crystalline Fe(III) forms that were
otherwise not reduced (Lovley et al. 1998). Soluble
quinones are probably more accessible to microorganisms than insoluble Fe(III) oxides and can thus establish
contact with Fe(III) oxides more easily than the cells
(Lovley 2000). Even in environments with low concentrations of humic substances, microbial electron transfer
to humics may still be significant. As long as Fe(III) is
present, humics can transfer electrons to the oxide and
thus be continually recycled; AQDS as low as 10 lM
can stimulate the reduction of Fe(III) oxides in aquifer
sediments (Lovley 2000).
Humic substances may also enhance Fe(III) reduction
through complexation of Fe(II) (Royer et al. 2002a, b).
Studies with Shewanella putrefaciens CN32 suggest that
addition of natural organic matter improved hematite
reduction via electron shuttling initially and, later, via
complexation mechanisms (Royer et al. 2002a). Fe(II)
complexation occurred only after sufficient Fe(II) had
accumulated in the system (Royer et al. 2002b). This
complexation likely improved Fe(III) reduction by
preventing Fe(II) sorption to hematite and cell surfaces.
Besides enhancing Fe(III) reduction, humic substances may also improve contaminant degradation by
increasing the bioavailability of poorly soluble pollutants. The addition of humic substances stimulates the
degradation of various polyaromatic hydrocarbons
(PAHs) under aerobic conditions (Ortega-Calvo &
Saiz-Jimenez 1998; Haderlein et al. 2001). The low
toxicity of humic substances makes them an attractive
alternative over synthetic surfactants which also improve bioavailability of polyaromatic hydrocarbons
(Tiehm 1994; Robertson 1998). However, the mechanisms by which humic substances increase PAH bioavailability are still unclear.
Y-Su Luu and J.A. Ramsay
acetate as both a carbon and energy source, and hence
require it at higher concentrations (Coates et al. 2002).
The environmental significance of humics oxidation has
yet to be quantified. However, this type of respiration is
believed to be important in environments low in Fe(III),
where reduced humics can diffuse into less reducing
zones and act as electron donors for the reduction of
nitrate and other electron acceptors (Lovley 2000).
Conclusions
The capacity for Fe(III) reduction is found in phylogenetically diverse microorganisms throughout the Bacteria and Archaea domains. However, the biochemistry
of microbial Fe(III) reduction is still a relatively new
area of study, with most work focusing on pure cultures
of Shewanella and Geobacter. Given the phylogenetic
diversity of dissimilatory Fe(III)-reducing bacteria and
the different environments they inhabit, it is likely that
mechanisms of Fe(III) reduction vary among different
groups of dissimilatory Fe(III)-reducing bacteria. In
Shewanella species alone, there are at least two pathways
for Fe(III) reduction involving either membrane-bound
proteins and cytochromes, or microbially secreted electron shuttles. In Geobacter, most evidence points to
membrane-bound ferric reductase activity, with direct
cell–oxide contact necessary in the absence of exogenous
electron shuttles. As more dissimilatory Fe(III)-reducing
bacteria are identified and studied, new mechanisms for
accessing and reducing Fe(III) will likely emerge.
In nature, the factors controlling the rate and extent
of Fe(III) are more complex than in pure culture studies.
Electron shuttling via humic substances may be an
important mechanism for accessing and reducing Fe(III)
in environments rich in organic matter. It is an energetic
advantage for dissimilatory Fe(III)-reducing bacteria
to use naturally present electron shuttling compounds
rather than produce their own. The ability of humic
substances to stimulate Fe(III) reduction may have
important environmental implications such as in bioremediation.
Humic substances as electron donors
In addition to acting as electron acceptors for microbial
respiration, humics can also serve as electron donors to
other bacteria. Microorganisms capable of oxidizing
humic substances are phylogenetically diverse and have
been isolated from a variety of environments (Coates
et al. 2002). Lovley et al. (1999) found that several
Fe(III)- and humics-reducing microorganisms are able
to oxidize reduced humics and/or anthrahydroquinone2,6-disulphonate with nitrate, fumarate, selenate or
arsenate as electron acceptors. These microorganisms
obtain carbon from sources such as acetate, and use
oxidized humics as an energy source (Coates et al.
2002). This ability may provide these organisms with a
competitive advantage over other bacteria that need
Acknowledgements
The authors acknowledge the financial support of
Environment Science and Technology Alliance Canada
(ESTAC) and the Natural Science and Engineering
Research Council of Canada (NSERC).
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