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Geomicrobiology Journal, 21:379–391, 2004
Copyright CTaylor & Francis Inc.
ISSN: 0149-0451 print / 1362-3087 online
DOI: 10.1080/01490450490485872
Potential for Microscale Bacterial Fe Redox Cycling
at the Aerobic-Anaerobic Interface
Eric E. Roden,1Dmitri Sobolev,1,2Brian Glazer,3and George W. Luther, III3
1Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487, USA
2University of Texas Marine Science Institute, Port Aransas, Texas 78373, USA
3College of Marine Studies, University of Delaware, Lewes, Delaware 19958, USA
Recent studies of bacterial Fe(II) oxidation at circumneutral pH
by a newly-isolated lithotrophic β-Proteobacterium (strain TW2)
are reviewed in relation to a conceptual model that accounts for
the influence of biogenic Fe(III)-binding ligands on patterns of
Fe(II) oxidation and Fe(III) oxide deposition in opposing gradi-
ents of Fe(II) and O2. The conceptual model envisions complexa-
tion of Fe(III) by biogenic ligands as mechanism which alters the
locus of Fe(III) oxide deposition relative to Fe(II) oxidation so as
to delay/retard cell encrustation with Fe(III) oxides. Experiments
examining the potential for bacterial Fe redox cycling in micro-
cosms containing ferrihydrite-coated sand and a coculture of a
lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissim-
ilatory Fe(III)-reducing bacterium (Shewanella algae strain BrY)
are described and interpreted in relation to an extended version
of the conceptual model in which Fe(III)-binding ligands promote
rapid microscale Fe redox cycling. The coculture systems showed
minimal Fe(III) oxide accumulation at the sand-water interface,
despite intensive O2input from the atmosphere and measurable
dissolved O2to a depth of 2 mm below the sand-water interface. In
contrast, a distinct layer of oxide precipitates formed in systems
containing Fe(III)-reducing bacteria alone. Voltammetric micro-
electrode measurements revealed much lower concentrations of
dissolved Fe(II) in the coculture systems. Examination of materials
from the cocultures by fluorescence in situ hybridization indicated
close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-
reducing bacteria in the upper few mm of sand. Together these
results indicate that Fe(II)-oxidizing bacteria have the potential to
enhance the coupling of Fe(II) oxidation and Fe(III) reduction at
redox interfaces, thereby promoting rapid microscale cycling of Fe.
Keywords chemolithotrophy, Fe(II) oxidation, Fe redox cycling,
metabolic energetics, microenvironment, microscale
Received 27 January 2004; accepted 14 May 2004.
This work was supported by awards from the U.S. National Sci-
ence Foundation, the U.S. Department of Energy, The University of
Alabama, School of Mines and Energy Development, and the Sigma
Xi Grant-in-Aid Research Program.
Address correspondence to Eric E. Roden, Department of Biologi-
cal Sciences, The University of Alabama, A122 Bevill Bldg. 7th Ave,
Tuscaloosa, Alabama 35487-0206, USA. E-mail: eroden@bsc.as.ua.
edu
INTRODUCTION
Bacterial Fe Redox Cycling
The redox cycling of iron (Fe) exerts a strong influence on
the behavior of various organic and inorganic compounds in
aquatic systems (Stumm and Sulzberger 1992; Davison 1993)
(see Figure 1). Ferric oxyhydroxides (e.g., Fe(OH)3, FeOOH,
Fe2O3) comprise the stable (and highly insoluble) form of Fe in
aerobic environments at circumneutral pH (Cornell and
Schwertmann 1996). Fe(III) oxides generally possess high sur-
face areas covered by reactive –OH functional groups, and are
thus important sorbents for a wide variety of organic and in-
organic contaminants in both open water and soil/sedimentary
environments (Stumm 1992). Fe(III) oxides are subject to nonre-
ductive dissolution as well as reductive dissolution under anaero-
bic conditions (Luther et al. 1992). When these processes occur,
Fe(II) and OH−, together with sorbed and coprecipitated species,
are released to solution through generalized reactions such
as
Fe(OH)3∼X+e−→Fe2++3OH−+X [1]
where X represents a sorbed species released to solution during
Fe(III) oxide dissolution. Direct microbial (enzymatic) reduc-
tion coupled to oxidation of organic carbon and H2is recog-
nized as the dominant mechanism for Fe(III) oxide reduction
in nonsulfidogenic anaerobic soils and sediments (see Lovley
1991, 2000 for review). This process contributes to both natu-
ral and contaminant (hydrocarbon) organic carbon oxidation in
sedimentary environments, and exerts a broad range of impacts
on the behavior of trace and contaminant metals and radionu-
clides (Lovley and Anderson 2000). Reduced sulfur (S) oxida-
tion processes (both biotic and abiotic) drive much of Fe(III) ox-
ide reduction in sulfur-rich marine sediments (e.g. Thamdrup,
Fossing, and Jorgensen 1994), although the potential for sub-
stantial organic carbon oxidation coupled to direct enzymatic
Fe(III) oxide reduction in marine sediments is now recognized
(Thamdrup 2000). Bacterial catalysis is also likely to play an
379
380 E. E. RODEN ET AL.
Figure 1. The role of Fe redox transformations in biogeochemical cycling. Modified with permission from Figure 1 in Tebo and
He (1999), copyright 2000 American Chemical Society. Recent reviews of the role of microbial activity in the various processes are
indicated by the superscripted letters: (a) circumneutral pH: Lovley (2000), Thamdrup (2000); acidic pH: Johnson (1998), Blake
and Johnson (2000), see also K¨usel et al. (1999) and Peine et al. (2000) (b) circumneutral pH: direct enzymatic catalysis doubtful;
see Thamdrup et al. (1993), Lovley (1994), and Schippers and Jørgensen (2001); acidic pH: Blake and Johnson (2000), Pronk and
Johnson (1992). (c) involvement of enzymatic catalysis not yet known. (d) circumneutral pH: Emerson (2000); acidic pH: Johnson
(1998), Blake and Johnson (2000); see also Edwards et al. (2000). (e, f) Straub et al. (2001).
important role in Fe(III) reduction coupled to oxidation of or-
ganic carbon and reduced S in acidic sedimentary environments
(Johnson, McGinness, and Ghauri 1993; Kusel et al. 1999; Kusel
and Dorsch 2000; Peine et al. 2000).
When Fe(II) comes into contact with O2or other suitable ox-
idants, Fe(II) can be reoxidized to soluble Fe(III), which even-
tually precipitates as Fe(III) oxyhydroxides (Taillefert, Bono,
and Luther 2000). The dominant role of microbial catalysis in
Fe(II) oxidation in acidic environments (e.g., acid mine drainage
and acid hot springs) is well-established (Brock and Gustafson
1972; Singer and Stumm 1972; Johnson et al. 1993). In contrast,
Fe(II) is subject to rapid chemical oxidation by dissolved O2at
circumneutral pH (Davison and Seed 1983; Millero, Sotolongo,
and Izaguirre 1987), and the Fe(III) produced eventually hy-
drolyzes and precipitates through overall reactions such as
Fe2++0.25O2+2.5H2O→Fe(OH)3+2H+
G◦=−109 kJ mol−1[2]
In view of the speed of this spontaneous abiotic reaction (the
half-life of dissolved Fe2+in air-saturated water is on the or-
der of a few minutes), bacterial Fe(II) oxidation with O2as
an electron acceptor at circumneutral pH, though energetically
more favorable than Fe(II) oxidation at low pH, has been con-
sidered doubtful from a geochemical perspective (e.g., Davison
and Seed 1983). Nevertheless, bacteria have been associated
with circumneutral Fe(II) oxidation and Fe(III) oxyhydroxide
deposition for a over a century (Ghiorse 1984; Ehrlich 1995).
Areview by Emerson (2000) provides an excellent overview
of the history of research on circumneutral bacterial Fe(II) ox-
idation, as well as the physiology and systematics of currently
known FeOB. It is relevant to note that the potential for both
anaerobic phototrophic (Widdel et al. 1993; Ehrenreich and
Widdel 1994) and chemolithotrophic nitrate-reducing bacteria
(Straub et al. 1996; Benz, Brune, and Schink 1998; Straub and
Buchholz-Cleven 1998) to catalyze circumneutral Fe(II) oxi-
dation is now recognized (Straub et al. 2001). However, the
overview and experimental studies presented in this paper fo-
cus on chemolithotrophic organisms capable of oxidizing Fe(II)
with O2as an electron acceptor.
Circumneutral Bacterial Fe(II) Oxidation
Recent enrichment and isolation studies with Fe(II)-O2op-
posing gradient systems (Kucera and Wolfe 1957; Jones 1983)
have expanded the range of organisms known to be involved
in aerobic circumneutral Fe(II) oxidation beyond the traditional
stalk-forming Gallionella and sheathed bacteria of Sphaerotilus-
Leptothrix group, to include unicellular organisms from the α-,
β-, and γ-Proteobacteria (Emerson and Moyer 1997; Emerson
2000; Edwards et al. 2003; Sobolev and Roden 2004). The in-
volvement of unicellular bacteria in circumneutral Fe(II) oxi-
dation is consistent with the observation of high numbers of
unicellular organisms closely associated with Fe(III) oxides in
POTENTIAL FOR MICROSCALE BACTERIAL Fe REDOX CYCLING 381
microbial mats present at a groundwater Fe seep in Denmark
(Emerson and Revsbech 1994a), and the Fe(III) oxide-rich
plaque of aquatic macrophyte roots (Emerson, Weiss, and
Megonigal 1999). Studies by Emerson and colleagues (Emerson
and Revsbech 1994b; Emerson and Moyer 1997; Neubauer,
Emerson, and Megonigal 2002) have clearly demonstrated the
potential for chemolithotrophic growth of such organisms cou-
pled to Fe(II) oxidation at circumneutral pH.
FeOB are likely to play a significant role in circumneu-
tral Fe(II) oxidation at redox interfacial environments where
diffusion-limited O2transport leads to low dissolved O2partial
pressure (microaerobic conditions) within the zone of Fe(II)-O2
overlap, i.e., environments characterized by opposing diffusion
gradients of O2and Fe(II). Such environments include stream
sediments, groundwater Fe seeps, wetland surface and rhizo-
sphere sediments, cave walls, irrigation ditches, subsurface bore-
holes, municipal and industrial water distribution systems, and
hydrothermal vents (Emerson 2000). Bacterial Fe(II) oxidation
may also occur in association with microscale redox zonation
within low-porosity matrix aggregates in subsurface sediments
(Hunter, Wang, and VanCappellen 1998).
In order to understand how bacterial Fe(II) oxidation can
compete effectively with chemical oxidation by O2under mi-
croaerobic conditions, it is useful to consider the kinetics vs. the
thermodynamics of Fe(II) oxidation by O2as a function of Fe(II)
and O2concentration at circumneutral pH (Figure 2). Assum-
ing a constant dissolved Fe(II) concentration of 100 µMatpH
7.0, Gcalculations (using thermodynamic data from Stumm
and Morgan 1996) indicate that the free energy associated with
Fe(II) oxidation (Equation 2) decreases only slightly (<5%)
as the dissolved O2concentration decreases from 300 µMto
1µM; in contrast, the half-life of Fe(II) (estimated using the
rate expression for abiotic Fe2+oxidation by O2given in Singer
and Stumm 1972) increases by a factor of 300. Thus, assuming
Figure 2. Change in the free energy yield (G)versus the
kinetics of abiotic dissolved Fe(II) oxidation as a function of
dissolved O2concentration. A constant pH of 7.0 and dissolved
Fe(II) concentration of 100 µMwas assumed for the calcula-
tions, and activities were assumed to be equal to concentrations.
Rates of abiotic Fe(II) oxidation used to compute half-lives (t1
/
2
values) for Fe(II) were computed using the rate expression given
in Singer and Stumm (1972).
FeOB can compete with abiotic Fe(II) oxidation under microaer-
obic conditions, considerable amounts of energy should still be
available for chemolithotrophic metabolism. Flow chamber ex-
periments have in fact demonstrated that FeOB can compete
effectively with abiotic Fe(II) oxidation under microaerobic con-
ditions (Emerson and Revsbech 1994b).
Potential for Coupling of Bacterial Fe(II) Oxidation
and Fe(III) Oxide Reduction in Natural Systems
Compared to reduced Mn, S, and N oxidation processes in
which the quantitative importance of enzymatic catalysis by
chemolithotrophic bacteria is well-recognized (Jorgensen 1989),
the significance of bacterial Fe(II) oxidation as a biogeochem-
ical process is still not well understood. The activity of FeOB
leads to the deposition of mainly amorphous Fe(III) oxide phases
which are excellent substrates for anaerobic dissimilatory FeRB
(Emerson and Revsbech 1994a). Since this deposition typically
takes place within redox interfacial environments characterized
by low dissolved O2concentration, the possibility exists that
bacterial Fe(II) oxidation and Fe(III) reduction are tightly cou-
pled in such environments (Emerson and Moyer 1997), at least
in freshwater systems where the impact of S cycling on Fe dy-
namics is much lower than in marine systems. This proposed
interaction is analogous to the well-known coupling between
bacterial sulfate reduction and lithotrophic or phototrophic re-
duced S oxidation in microbial mats and marine environments
(Jorgensen 1989).
Emerson and Moyer (1997) found that FeOB do not al-
ter the rate of Fe(III) oxide accumulation in circumneutral pH
diffusion-controlled opposing-gradient culture systems, a result
verified by our own studies with opposing-gradient FeOB cul-
tures (Sobolev and Roden 2004). In addition, Neubauer et al.
(2002) showed that FeOB only slightly enhanced (18%) total
rates of Fe(II) oxidation over cell-free controls in well-mixed
batch reactors. This situation differs from radically from oxida-
tion of Fe(II) at low pH and oxidation of dissolved sulfide at
neutral pH, the kinetics of which may be accelerated by a factor
of 1,000 or more in the presence of organisms such as Thiobacil-
lus (Singer and Stumm 1972) and Beggiatoa (Jorgensen and
Revsbech 1983; Nelson, Jorgensen, and Revsbech 1986), re-
spectively. Together these results suggest that the unique role of
FeOB in Fe redox cycling in nature is likely to stem from their
ability to alter the spatial separation between zones of Fe(II) ox-
idation and Fe(III) oxide precipitation in a way that promotes Fe
redox cycling. It is important to note in this context that FeOB
activity generally leads to the development of a narrow band
of cells and Fe(III) oxides in the vicinity of aerobic/anaerobic
interface in Fe(II)-O2opposing gradient systems (Emerson and
Moyer 1997; Benz et al. 1998); see also Figure 3), in contrast to
a much more diffuse zone of Fe(III) oxide deposition in abiotic
systems. These observations suggest that FeOB have the capa-
bility to focus the zone of Fe(III) oxide deposition very close
to (i.e. within a few mm) the zone where FeRB are expected to
be active. This capability is significant in light of the fact that
382 E. E. RODEN ET AL.
Figure 3. Distribution of Fe(II), particulate Fe(III) oxides, O2,
and bacteria in 1-week old sterile (A) and TW2-inoculated (B)
opposing-gradient cultures. Note that Fe(II) profiles determined
by densitometric analysis of the ferricyanide-fixed diffusion
probes were confounded by the presence of Fe(III) compounds
in the probe (see text). Fe(II), O2, and bacterial profiles are av-
erages of measurements in triplicate cultures. Reproduced with
permission from Sobolev and Roden (2001), copyright 2001
American Society for Microbiology.
the presence of even traces of dissolved O2are likely to either
inhibit strictly anaerobic FeRB metabolism (Lovley 2002), or
strongly depress Fe(III) reduction activity in facultative FeRB
(Arnold et al. 1990). FeOB stand to benefit greatly from alter-
ing the geometry of the Fe(II)-O2reaction zone such that little
or no spatial separation exists between the zones Fe(III) oxide
deposition and Fe(II) regeneration (discussed in detail later).
The following sections of this paper provide a review of
our recent studies on FeOB metabolism and associated geo-
chemical phenomena in Fe(II)-O2opposing gradient systems,
and on the potential for coupled FeOB/FeRB metabolism in
such systems. Although much of the reviewed research has al-
ready been published (Sobolev and Roden 2001, 2002, 2004),
several previously undescribed aspects of our studies are con-
sidered here, including calculations of the energetics of FeOB
metabolism in relation to production of Fe(III)-binding ligands,
and microelectrode measurements of dissolved Fe(II) and O2
distributions in FeOB/FeRB cocultures. These studies are dis-
cussed and interpreted in relation to conceptual models which
account for the influence of biogenic Fe(III)-binding ligands on
patterns of Fe(III) oxide deposition and microbially-catalyzed
Fe redox cycling in opposing gradients of Fe(II) and O2.
NOVEL EFFECTS OF FeOB ACTIVITY ON Fe(II)
OXIDATION AND Fe(III) OXIDE DEPOSITION
We have recently conducted a series of experimental studies
to evaluate quantitative aspects of bacterial Fe(II) oxidation and
its potential consequence for Fe cycling in redox interfacial en-
vironments. This work was conducted with a chemolithotrophic
Fe(II)-oxidizing bacterium isolated from a freshwater wetland in
the Talladega National Forest in northcentral Alabama, United
States (Sobolev and Roden 2001). This organism, designated
strain TW2, falls within the β-subclass of the Proteobacteria
with 91% 16S rRNA gene sequence identity with G. ferrug-
inea (Sobolev and Roden 2004). The sediments in the Talladega
Wetland (TW) from which strain TW2 was isolated are typical
of organic-rich freshwater wetlands (Westermann 1993), char-
acterized by steep gradients of dissolved O2and high concen-
trations of dissolved and solid-phase Fe(II) within mm of the
sediment-water interface (Roden and Wetzel 1996; Sobolev and
Roden 2002). It is important to acknowledge that our findings
with strain TW2 may not necessarily apply to other phyloge-
netically and physiologically different neutrophilic FeOB, in
particular the unicellular FeOB from the γ-subclass of the Pro-
teobacteria that have been isolated from a variety of environ-
ments by Emerson and colleagues (Emerson and Moyer 1997;
Emerson et al. 1999; Emerson and Moyer 2002; Neubauer et al.
2002; Weiss et al. 2003). For example, the ability of the latter
organisms to alter geochemical conditions at the Fe(II)-O2inter-
face through production of soluble/colloidal Fe(III) compounds,
and/or to grow in close association with FeRB, has not yet been
evaluated. In addition, the extent to which the results obtained in
our laboratory pure culture systems reflect what occurs in nature
is unknown. Hence, the studies discussed here should be viewed
as a starting point for future evaluations of the unique physio-
logical and biogeochemical activities of FeOB in circumneutral
sedimentary environments.
Opposing-Gradient Studies
Studies of the growth of strain TW2 in Fe(II)-O2opposing
gradient cultures have revealed several novel effects of bacterial
metabolism on the dynamics of Fe(II) oxidation and Fe(III) ox-
ide deposition at circumneutral pH (Sobolev and Roden 2001).
The opposing-gradient culture systems used in these studies
were analogous to those employed by Emerson and Moyer
(1997), except that a relatively high concentration of soluble
Fe(II) (50 mM FeCl2)was employed in the bottom layer in or-
der to stimulate FeOB growth and better observe the effects of
FeOB activity on patterns of Fe(III) oxide deposition. Dissolved
Fe(II) concentrations were likely on the order of several hundred
POTENTIAL FOR MICROSCALE BACTERIAL Fe REDOX CYCLING 383
µMinthe zone of Fe(II) oxidation a few cm above the bottom
layer (see Figure 5). These reaction systems mimic Fe-rich sur-
face sediments in freshwater wetlands (e.g., Roden and Wetzel
(1996)), as well as certain highly reducing groundwater environ-
ments (e.g., the landfill leachate-contaminated aquifers studied
by Christensen et al. 1994) in which porefluids containing 10s
to 100s of micromoles of dissolved Fe(II) per L may impinge
on downstream aerobic groundwaters.
Oxygen gradients were much steeper in culture systems in-
oculated with FeOB than in sterile controls (Figure 3), similar
to observations made by Emerson and Moyer (1997) and more
recently by Edwards et al. (2003). Analysis of microscope slides
embedded in the agar-stabilized cultures, to which FeOB became
attached during a 1-week growth period, revealed a peak in bac-
terial biomass at a depth where the dissolved O2concentration
was less than 20% saturation at room temperature (<50 µM)
(Sobolev and Roden 2001). An interesting observation emerged
during recording of the O2microprofiles in inoculated vs. sterile
cultures: the lower boundary of the zone of Fe(III) oxide depo-
sition (Figure 4, hatched bars), defined by the depth at which
the tip of the O2microelectrode became visible after exiting the
oxide layer, was consistently below the depth of O2penetration
in the inoculated cultures. In contrast, the entire zone of oxide
deposition was aerobic in the abiotic controls. Time course mea-
surements of O2distributions indicated that the observed suboxic
deposition of Fe(III) oxides in the live cultures was not due to an
upward-retreating O2front, because the depth of O2penetration
Figure 4. Time course measurements of dissolved O2micro-
profiles in Fe(II)-opposing gradient cultures in the presence (A)
and absence (B) of FeOB.
increased steadily over time as a result of progressive depletion
of soluble Fe(II) in the bottom layer of the cultures (Figure 4).
In addition, the depth of O2penetration was above the bottom
boundary of the oxide band at all times except for the first two
time points in inoculated cultures, whereas O2always penetrated
to depths below the lower boundary of the oxide band in sterile
controls (Sobolev and Roden 2001). As discussed further below,
these findings provide evidence for the ability of FeOB activity
to alter patterns of Fe(III) oxide deposition in a way that would
be conducive to coupling of bacterial Fe(II) oxidation and Fe(III)
reduction, since Fe(III) compounds deposited below the depth
of O2penetration would be immediately available as electron
acceptors for FeRB.
Thin film diffusion probes (see Figure 5) were deployed in
an attempt to assess the influence of FeOB on Fe(II) microgradi-
ents at the aerobic/anaerobic interface, in a manner analogous to
how S2−microelectrodes have been used to quantify the effect
of chemolithotrophic S-oxidizing bacteria on dissolved sulfide
microgradients (Jorgensen and Revsbech 1983; Nelson et al.
1986). Unfortunately, because of interference by Fe(III) with
the densitometric measurements, the diffusion probes did not
Figure 5. Photos of ferricyanide-fixed thin-film diffusion
probes from control (left) and TW2-inoculated (right) oppos-
ing gradient cultures shown in Figure 4. Arrows indicate the
depth of O2penetration (zO2) and the location of the bacterial
plate (zBact). Note that heavy Fe(III) deposits in the probe from
the control culture (partially evident as a grayish band gray just
above zO2) were obscured by the presence of Fe(II) in the probe.
The bottom bars are results from probes submerged overnight in
standard Fe(II)-EDTA and FeCl3solutions. The standards were
photographed under different light conditions and are not di-
rectly comparable with probes from the cultures. Modified with
permission from Sobolev and Roden (2001), copyright 2001
American Society for Microbiology.
384 E. E. RODEN ET AL.
provide accurate measurements of dissolved Fe(II) microgradi-
ents at the aerobic/anaerobic interface. However, they revealed
two previously undescribed effects of FeOB activity on circum-
neutral Fe(II) oxidation, which are described next.
Quantitative Contribution of FeOB to Fe(II) Oxidation
Time-course studies have shown that bulk rates of Fe(III) ox-
ide deposition are similar in abiotic control and FeOB-inoculated
opposing-gradient cultures (Emerson and Moyer 1997; Sobolev
and Roden 2004). In contrast, visual analysis of diffusion probes
emplaced in TW2 cultures (Figure 5) revealed the presence of
much heavier Fe(III) oxide deposits in probes from control cul-
tures compared to those from inoculated ones. In order to ver-
ify quantitatively the lower abundance of particulate oxides in
probes from the live cultures, the concentration of Fe(III) in
probes (determined by scraping the film off the glass slide, fol-
lowed by extraction in 0.5 M HCl and Ferrozine analysis) as well
as samples of whole medium were measured in replicate (n =3)
live and control cultures. When corrected for the presence of sol-
uble/colloidal Fe(III) (see Figure 6B and discussion below), the
Figure 6. Fe(II) and Fe(III) in whole medium samples and
diffusion probes from control and TW2-inoculated opposing-
gradient cultures. (A) Comparison among probes and whole
medium. The smaller shaded bar superimposed on the Fe(III)
bar for live culture probes represents the estimated particulate
Fe(III) concentration after correction for the presence of soluble
Fe(III). (B) Comparison between washed and unwashed probes
(see text). Data represent averages of triplicate cultures; error
bars show 95% confidence interval. Reproduced with permis-
sion from Sobolev and Roden (2001), copyright 2001 American
Society for Microbiology.
estimated concentration of particulate Fe(III) in probes from the
live cultures was ca. 4-fold lower than in probes from the abiotic
controls (Figure 6A). In addition, the concentration of Fe(III)
was ca. 3-fold lower in probes compared to whole medium in
live cultures, whereas probe and whole medium Fe(III) concen-
trations were nearly identical in abiotic systems (Figure 6A).
These findings confirmed the consistent visual observation of
lower Fe(III) oxide abundance in probes from live vs. control
cultures.
The relative scarcity of particulate oxides in diffusion probes
from the live cultures suggests that Fe(II) oxidation was domi-
nated by bacterial activity. Because the 5% agar content of the
probe would be expected to effectively exclude bacteria, only
chemical oxidation is expected to have occurred within the agar
film. The low abundance of oxide deposits in probes from the
live cultures therefore suggests that FeOB activity in the bulk
medium scavenged Fe(II) rapidly enough to strongly depress
Fe(II) diffusion into the probe and subsequent abiotic oxidation.
Mass balance calculations on the Fe content of probes vs. bulk
medium, which accounted for the presence of soluble Fe(III) in
probes from live cultures (discussed next), indicated that bacte-
rial activity could account for ca. 90% of Fe(II) oxidation occur-
ring in the live cultures (Sobolev and Roden 2001). This fraction
might be higher in steady-state systems, since some Fe(II) oxi-
dation inevitably occurred abiotically before the FeOB plate was
established in the live cultures. These results demonstrate that
FeOB can compete successfully with abiotic Fe(II) oxidation in
diffusion-limited opposing-gradient systems.
Production of Soluble or Colloidal Fe(III) Compounds:
Experimental Evidence and Theoretical Considerations
A second important observation from the thin-film diffusion
probes was the presence of a green band in ferricyanide-treated
probes from the live cultures (Figure 5, top right). The green band
was likely due to the presence of soluble or colloidal Fe(III) com-
pounds which reacted with ferricyanide reagent (note that ferri-
cyanide forms a bright blue precipitate with Fe(II); see Sobolev
and Roden (2001) for further details on the use of ferricyanide
with the diffusion probes). Ferricyanide was shown to form a
green precipitate with Fe(III) at neutral pH (Figure 5, standards),
and no other components in the media were present in quanti-
ties sufficient to produce such a colored precipitate. Since green
bands were not evident in the control cultures, the formation of
soluble or colloidal Fe(III) can be attributed to FeOB activity.
Washing of nonferricyanide-treated probes (by repeated dipping
in anaerobic distilled water inside an anaerobic chamber) from
the live culture resulted in removal of approximately 70% of
their Fe(III) content (Figure 6B, right); in contrast, identical
treatment of probes from the abiotic control cultures resulted
in no change in Fe(III) content, whereas the amount of Fe(II)
was significantly decreased (Figure 6B, left). These results ver-
ified the presence of soluble/colloidal Fe(III) in probes from the
live cultures, and further indicated that the presence of mobile
POTENTIAL FOR MICROSCALE BACTERIAL Fe REDOX CYCLING 385
Fe(III) is a bacterially mediated phenomenon in these cultures.
pH decrease associated with bacterial Fe(II) oxidation at O2-
Fe(II) boundary could potentially account for Fe(III) remaining
in solution. However, in our experiments such a decrease was far
less than would be required to stabilize any significant amount
of Fe(III), as the lowest pH value observed (with a 1-mm diam-
eter pH minielectrode; Orion Instruments) was 6.6 in the zone
of oxide deposition, compared to 7.2 at the surface.
It is possible that Fe(III) was kept in the solution in the
live cultures by metal-binding ligands excreted by the bacteria
specifically for the purpose of retarding cell surface encrustation
with oxide precipitates. Although speculative, this suggestion
is consistent with the fact that encrustation of the FeOB cells
with particulate oxides, leading to their eventual entombment,
is likely to be one of the major environmental challenges faced
by gradient-dwelling, solid-phase oxide-producing microorgan-
isms (Emerson and Moyer 1997). Formation of soluble or col-
loidal Fe(III) compounds and eventual remote deposition of the
oxides may reduce or delay such encrustation. This suggestion
is supported by the observation that certain phototrophic FeOB
have been shown to avoid cell encrustation with Fe(III) ox-
ides while accumulating significant amounts of soluble Fe(III);
in contrast, self-encrusting organisms did not form significant
amounts of soluble Fe(III) (Straub et al. 2001). We hypothesize
that, as soluble or colloidal Fe(III) complexes diffuse away from
the locus of Fe(II)-oxidation activity, they become destabilized,
resulting in precipitation of Fe(III) oxides both within and below
the zone of oxygen penetration (Figure 7). This process could
account for the suboxic deposition of Fe(III) oxides observed in
FeOB-inoculated cultures (Figure 3B).
The validity of the above hypothesis requires that neutrophilic
FeOB gain sufficient energy from Fe(II) oxidation to support
the synthesis of relatively large quantities of Fe(III)-binding
ligands. In order to evaluate this assumption quantitatively, a
metabolic energetics analysis was conducted following the ap-
proach described in Rittman and McCarty (2001). The analysis
involves comparison of the amount of energy required to con-
vert a carbon source to cellular carbon with the amount of energy
Figure 7. Conceptual model for suboxic Fe(III) oxide deposi-
tion coupled to production of soluble Fe(III)-ligand complexes
by FeOB.
liberated from energy-generating reactions, taking into account
the efficiency of cellular energy transfer. Following Rittman and
McCarty (2001), we assume that the carbon source (here dis-
solved HCO−
3)isconverted to pyruvate as a representative in-
tracellular intermediate. The energy required for this conversion
(referred to as Gp, which is equal to the G◦ of the reaction
assuming standard conditions at pH 7) was estimated from the
following reaction:
0.3HCO−
3+Fe2++2.4H2O→0.1CH3COCOO−+Fe(OH)3
+1.8H+G◦ =+5.8kJ/e−[3]
in which the electrons required for carbon fixation are obtained
from oxidation of Fe(II) to Fe(OH)3(amorphous Fe(III) oxyhy-
droxide). The free energies of formation required for this and
other energetics calculations were obtained from Stumm and
Morgan (1996) and Thauer, Jungermann, and Decker (1977). An
energy requirement of +18.8 kJ/e−was assumed for conversion
of pyruvate to cellular carbon (Gpc), based on a value of 3.33 kJ
per gram cells with NH+
4as the nitrogen source, and a standard
cellular composition of C5H7O2N (McCarty 1971). This stoi-
chiometry corresponds to 5.65 g cells per electron equivalent
assuming the following biosynthesis reaction:
0.05NH+
4+0.25HCO−
3+1.2H++e−→0.05C5H7O2N
+0.65H2O [4]
The total energy required for cellular biosynthesis (Gs)isthen
obtained from the equation
Gs=Gp/ε +Gpc/ε [5]
where εis the energy transfer efficiency. A standard εvalue
of 0.6 (Rittmann and McCarty 2001) was used in the calcula-
tions presented here, which produces a value of +41.0 kJ/e−
for Gs.Next, the overall amount of energy available for cel-
lular metabolism through Fe(II) oxidation (Gr, equal to G◦
assuming standard conditions at pH 7) was estimated from the
reaction
Fe2++0.25O2(aq) +2.5H2O→Fe(OH)3(s) +2H+
G◦ =−111.3kJ/e−[6]
Assuming that the transfer efficiency for this reaction is the
same as for biosynthesis reactions, the actual amount of energy
available is εGr=−66.8 kJ/e−. Finally, an energy balance is
defined in which oxidation of Aequivalents of electron donor
are required to supply energy for cell synthesis:
AεGr+Gs=0 [7]
According to equation 7, the fraction of total electron equiv-
alents devoted to cell synthesis (f0
sin Rittmann and McCarty
2001) is 1/(1 +A), which is equal to 0.613 using the Gp,
386 E. E. RODEN ET AL.
Gpc,Gr, and εvalues given before. With this value for f0
s,
the stoichiometry in equation 4, and the fact that one electron
equivalent is liberated per mol of Fe(II) oxidized, the estimated
the biomass yield (not accounting for maintenance energy or
endogenous decay) for FeOB growth is
0.613 ×0.25 mol cell-C/e−equiv ×1e−equiv/mol Fe(II)
=0.153 mol cell-C/mol Fe(II).
This yield is several-fold in excess of the observed net growth
yield for TW2 in opposing gradient cultures (YNet, equal to ca.
0.02 mol cell-C/mol Fe(II); Sobolev and Roden 2004). These
results suggest that a substantial amount of energy is available
for synthesis of other cellular components, e.g., Fe(III)-binding
ligands, by neutrophilic FeOB. They also clearly illustrate the
major difference in potential growth yield for neutrophilic vs.
acidophilic FeOB: analogous calculations in which Fe(II) was
assumed oxidize to soluble Fe3+at pH 2 produced growth yield
estimates on the order of 0.03 mol cell-C/mol Fe(II), in line
with both early (e.g., Silverman and Lundgren 1959) and more
recent (e.g., Harvey and Crundwell 1997) published values for
Thiobacillus ferrooxidans.
In order to evaluate the potential significance of nonstandard
conditions on the amount of energy that may be available for
synthesis of Fe(III)-binding ligands, a range of dissolved O2
and Fe(II) concentrations in the zone of Fe(II) oxidation (10–
100 µM and 100–1000 µM, respectively) was estimated based
on the microelectrode (Figure 3) and diffusion probe (Figure 5)
measurements. Although the estimated range of concentrations
is only a rough approximation, the calculations indicated (con-
sistent with the results shown in Figure 2) that the energetics of
circumneutral Fe(II) oxidation are not highly sensitive to dis-
solved O2and Fe(II) concentrations. Revised growth yields of
0.137–0.139 mol cell-C/mol Fe(II) were obtained by pairing
the lowest assumed dissolved O2concentration (10 µM) with
the highest dissolved Fe(II) concentration (1000 µM) and vice-
versa. If we assume a maximum total yield (YTotal)of0.138 mol
cell-C/mol Fe(II), then the potential yield for carbon allocation
to ligand biosynthesis (YLigand) can be estimated as follows:
YLigand =YTotal −YNet −bXt/Rt[8]
where YNet is the observed growth yield, bis a coefficient ac-
counting for endogenous cellular decay, and Xtand Rtare the
biomass (mol C/L) and rate of Fe(II) oxidation (mol/L/d) at time
t. The last term on the right-hand side of equation 8 accounts for
the influence endogenous decay on growth yield (Rittmann and
McCarty 2001). A value of 0.02 mol cell-C/mol Fe(II) was as-
sumed for YNet based on measured TW2 growth yields (Sobolev
and Roden 2004). A value of 0.05 d−1was assumed for b,
which is appropriate for relatively slow-growing microorgan-
isms (Rittmann and McCarty 2001). Xtwas estimated at ca. 1 ×
10−4mol C/L based on maximum cell densities obtained in op-
posing gradient cultures (Sobolev and Roden 2004), assuming
that all the cells in the cultures were contained in a ca. 2 mm
thick band in the culture tubes. Rtwas estimated at ca. 1 ×10−3
mol Fe(II)/L/d, using the pH-dependent second-order rate equa-
tion given in Singer and Stumm (1972), assuming a pH of 7 and
the range of dissolved O2and Fe(II) concentrations mentioned
previously. Note that use of this equation, which was originally
developed to predict pH-dependent rates of abiotic Fe(II) ox-
idation, to estimate rates of biotic Fe(II) oxidation implicitly
assumes that biotic oxidation was limited by O2and Fe(II) abun-
dance, and that all Fe(II) oxidation was biologically catalyzed.
Both of these assumptions are consistent with our studies of
strain TW2 in opposing gradient cultures (Sobolev and Roden
2001; Sobolev and Roden 2004). Plugging the estimated YTotal,
Ynet,b,X
t, and Rtvalues into equation 8 yielded a YLigand value
of 0.113 mol ligand-C/mol Fe(II). Multiplying this value by the
estimated rate of Fe(II) oxidation in turn yielded a value of ca.
1×10−4mol ligand-C L−1d−1for the rate of ligand biosynthe-
sis. At this rate of ligand biosynthesis, ligand concentrations on
the order of 1 mmol C L−1could be expected to accumulate in
the vicinity of the growth band after 1–2 week reaction period.
Although the capacity for biogenic ligands to complex or oth-
erwise stabilize Fe(III) (e.g., through the formation of polynu-
clear species with nm-scale hydrodynamic radii; vonGunten and
Schneider 1991; Taillefert et al. 2000) is unknown, the ener-
getic and biosynthesis calculations clearly suggest that accumu-
lation of significant quantities of extracellular ligands is pos-
sible in conjunction with FeOB activity in diffusion-controlled
opposing-gradient systems. The formation of such compounds
has important implications for the coupling of Fe oxidation and
reduction at redox interface environments. Such compounds, as
well as the reactive Fe(III) oxides generated upon their desta-
bilization, would be readily available for dissimilatory Fe(III)
reduction just below the depth of O2penetration. This phe-
nomenon represents unique mechanism whereby FeOB activ-
ity may alter the Fe(II)-O2reaction system so as to generate
conditions favorable for rapid microscale Fe redox cycling (see
below). This process would reduce the diffusion distance (travel
time) between sites of Fe(II) consumption and regeneration, and
alleviate the possible negative influence of Fe(II) sorption and/or
precipitation reactions on delivery of Fe(II) to FeOB.
EXPERIMENTAL STUDIES OF COUPLED BACTERIAL
Fe(II) OXIDATION AND Fe(III) REDUCTION
Apreliminary investigation of the potential for coupled Fe(II)
oxidation and Fe(III) reduction was conducted in experimental
microcosms in the presence and absence of strain TW2 (Sobolev
and Roden 2002). Based on the ability of TW2 to cause unique
alterations in patterns of Fe(III) oxide deposition in opposing
gradients of Fe(II) and O2,wehypothesized that this organism
would lead to enhanced coupling of Fe redox cycling at the
aerobic-anaerobic interface of the microcosms. The design and
results of these experiments, supported by previously unpub-
lished voltammetric microelectrode measurements of dissolved
POTENTIAL FOR MICROSCALE BACTERIAL Fe REDOX CYCLING 387
O2and Fe(II) distributions in the microcosms, are summarized
next. The findings are subsequently interpreted in relation to a
conceptual model of bacterially catalyzed Fe redox cycling at
the aerobic-anaerobic interface.
Coculture Systems
Strain TW2 was grown in opposing-gradient cultures as de-
scribed previously (Sobolev and Roden 2001), and the oxide-
rich band containing bacterial cells was collected from a 7–14 d
culture and used as the inoculum for the experiments described
next. Shewanella algae strain BrY, a facultative FeRB (Caccavo,
Blakemore, and Lovley 1992), was grown for 16 hr in tryptic
soy broth (TSB), harvested by centrifugation, washed (3X) with
Pipes buffer (10 mM, pH 7), and resuspended in anaerobic Pipes
buffer supplemented with Na-formate (10 mM) and vitamins and
minerals (Lovley and Phillips 1986). This cell suspension was
used as the inoculum for the coculture systems. Although strain
TW2 is capable of aerobic heterotrophic growth with acetate
as a carbon and energy source, it is not able to utilize formate
(Sobolev and Roden 2004). Hence, heterotrophic growth and
O2consumption by strain TW2 did not occur in the coculture
systems.
Amorphous Fe(III) oxide-coated sand was prepared by mix-
ing 75 mL of a suspension of hydrous ferric oxide gel (ca.
500 mmol Fe(III) L−1, prepared by neutralization of FeCl3as
described in Lovley and Phillips 1986), with 300 g of quartz
sand, −50 +70 mesh (Sigma Chemicals). The mixture was shell-
frozen in an alcohol bath and freeze-dried. The resulting oxide-
coated sand had a 0.5M HCl-extractable Fe(III) content of ca.
125 µmol g−1.
Bacterial Fe cycling microcosms were prepared be adding
40 g of the oxide-coated sand to 50 mL beakers (50 mm tall,
35 mm ID), to which 15 mL of the S. algae cell suspension was
added, resulting in ca. 1 mm of liquid over the sand surface.
Rossi-Cholodny buried slides for visualization/enumeration of
attached bacterial were then inserted. The cultures were incu-
bated in an anaerobic chamber (2–3% H2, balance N2) for 7 days,
after which they were removed from the chamber and inoculated
with the TW2 by evenly distributing the cell/oxide mixture over
the sand-liquid interface. A set of cultures not inoculated with
FeOB served as control. In addition, a set of cultures was killed
with 1 mM Na-azide (final concentration) to provide abiotic con-
trols. The cultures were covered loosely with aluminum foil and
allowed to sit open to the air on the laboratory bench.
After 2–3 d of incubation, the presence of Fe(III) oxide de-
posits on the surface of the microcosms was assessed visu-
ally and photographed. Oxygen microprofiles were recorded
with a Clark-style oxygen microelectrode as described previ-
ously (Sobolev and Roden 2001). In addition, microprofiles of
dissolved O2and Fe(II) were obtained simultaneously using a
gold amalgam voltammetric microelectrode (Brendel and Luther
1995). A detailed description of the methodology employed in
these measurements is provided in Brendel and Luther (1995)
and Luther et al. (1999). Rossi-Cholodny slides were retrieved
from the cultures and hybridized with a Shewanella-specific 16S
rRNA probe under conditions described previously (DiChristina
and Delong 1993). The Shewanella probe was labeled with the
fluorochrome Oregon Green (Molecular Probes, Inc.). The slides
were then counter-stained with DAPI and visualized under an
epifluorescent microscope with UV/visible light illumination.
EXPERIMENTAL RESULTS
Reduction of amorphous Fe(III) oxide by S. algae caused a
blackening of the oxide-coated sand (see Figure 8), presumably
due to production of magnetite or other mixed Fe(II)-Fe(III)
solid-phases (Fredrickson et al. 1998). Visual inspection of mi-
crocosms 2–3 d after inoculation with TW2 indicated a paucity
of surface Fe(III) oxide deposits in the cocultures (Figure 8A,
left). These results suggest that rates of Fe(II) oxidation and
Fe(III) reduction were approximately in balance within the up-
per few mm of the coculture systems, such that very little Fe(III)
oxide deposition took place. In contrast, controls lacking FeOB
(Figure 8A, right) or killed with azide (not shown) accumulated
an approximately 2 mm-thick oxide layer at the surface. Similar
results were observed in triplicate cultures in this and several
other experiments. No obvious difference in O2penetration or
patterns of oxide deposition were detected between the cultures
lacking FeOB and the azide-killed controls, which suggests that
O2scavenging due to aerobic respiration of S. algae did not exert
a significant influence on Fe(III) oxide deposition and inferred
Fe cycling in the cocultures (Sobolev and Roden 2002). Voltam-
metric microelectrode measurements revealed much lower con-
centrations of dissolved Fe(II) in cocultures compared to those
containing S. algae only (Figure 9). Fluorescence in situ hy-
bridization (FISH) analysis of buried slides from the coculture
microcosms indicated that FeOB and FeRB were found in close
proximity to one another within the aerobic zone (Figure 8B),
frequently in tight clusters with S. algae generally concentrated
towards the center of the clusters and TW2 on the periphery.
These findings suggest intimate involvement of FeOB in Fe re-
cycling processes within the microcosms.
Conceptual Model for Microscale Bacterial Fe Redox
Cycling
A conceptual model is provided in Figure 10 to explain the
differences in surface Fe(III) oxide accumulation in Fe(III)-
reducing microcosms with and without FeOB activity. Close
juxtapositioning of FeOB and FeRB and rapid microscale Fe
redox cycling within the Fe(II)-O2reaction zone in the cocul-
tures is hypothesized to maintain the majority of the Fe in the
reduced state at the sand-water interface, despite the presence of
detectable O2in the bulk aqueous phase. The observed clump-
ing of FeOB and FeRB around Fe(III) oxide aggregates suggests
that bacterial Fe(II) oxidation and Fe(III) reduction activities are
likely to have coexisted within the same horizon, with the FeRB
taking advantage of anaerobic microzones within the aggregates.
388 E. E. RODEN ET AL.
Figure 8. (A) Photo of bacterial Fe cycling microcosms containing both S. algae and TW2 (left) or S. algae only (right). Note
paucity of Fe(III) oxide surface precipitates in the coculture system. (B) FISH image of buried slide (ca. 1.5 mm depth) from a
coculture system hybridized with a Shewanella-specific 16S rRNA probe and counter-stained with DAPI. S. algae cells appear
yellow, TW2 cells appear blue. Arrow indicates TW2 cells surrounding an aggregate of amorphous Fe(III) oxide. Reproduced with
permission from Sobolev and Roden (2002), copyright 2002 Kluwer Academic Publishers.
These microzones are postulated to be generated by O2scaveng-
ing via Fe(II) oxidation by FeOB on the periphery of the aggre-
gates, leading to the development of “ultramicrogradients” of
O2at the surfaces of aggregates, with O2declining from the
bulk aqueous phase concentration to essentially zero at some
distance from the surface (Figure 10A, lower left). The lower
concentration of dissolved Fe(II) in microcosms inoculated with
TW2 compared to those containing S. algae only (Figure 9)
provides evidence for enzymatically enhanced Fe(II)/O2scav-
enging. As a result of these interactions, anaerobic conditions
would be maintained at the aggregate surface, allowing Fe(III)
reduction to proceed within a bulk aerobic environment. Pro-
duction and inward flux of biogenic ligand bound-Fe(III) would
presumably facilitate rapid Fe cycling at the aggregate surface.
With Fe trapped in a cycle between FeOB and FeRB, little or
no net oxide deposition would occur. In the absence of FeOB
activity, a lower degree of O2scavenging would allow O2to con-
tact aggregate surfaces (Figure 10B, lower left), where Fe(III)
reduction would thus be inhibited, leading to the accumulation
of oxide precipitates.
The above conceptual model, though speculative, is consis-
tent with existing models of the potential for anaerobic bac-
terial respiration within anoxic microniches in otherwise oxi-
dized aquatic sediments (Jorgensen 1977; Jahnke 1985). It is also
consistent with the recent demonstration of syntrophic growth
of sulfate-reducing and colorless sulfide-oxidizing bacteria
POTENTIAL FOR MICROSCALE BACTERIAL Fe REDOX CYCLING 389
Figure 9. Voltammetric microelectrode profiles of dissolved
O2and Fe(II) in Fe cycling microcosms containing both S. algae
and TW2 (A) or S. algae only (B). Data are averages of triplicate
profiles.
coupled to S redox cycling in O2-limited reaction systems
(vandenEnde, Meier, and vanGermerden 1997), in which O2
scavenging by the sulfide-oxidizer (Thiobacillus thioparus) al-
lowed the sulfate-reducer (Desulfovibrio desulfuricans)tore-
main active (mainly through reduction of highly reactive elemen-
tal S produced during sulfide oxidation) despite the continuous
input of dissolved O2. More detailed studies of the distribution
of FeOB and FeRB within Fe cycling aggregates, as well as
Figure 10. Conceptual model of Fe redox cycling in Fe(III)-
reducing microcosms with (A) and without (B) strain TW2.
Fe(OH)3refers to an aggregate of amorphous Fe(III) oxide.
Reproduced with permission from Sobolev and Roden (2002),
copyright 2002 Kluwer Academic Publishers.
more rigorous quantification of dissolved and solid-phase Fe(II)
and Fe(III) pools at the redox interface, are required to verify
this model for the behavior of our coculture systems. Develop-
ment and application of such techniques may also shed light
on the potential for bacterially catalyzed Fe redox cycling in
natural sedimentary environments. Preliminary studies in TW
surface sediments indicate the presence of comparable numbers
of FeOB and FeRB, as well as the presence of soluble Fe(III) at
the aerobic-anaerobic interface (Sobolev and Roden 2002). The
latter finding is consistent with other recent studies (employing
voltammetric electrodes as well as fine scale sectioning tech-
niques) which have demonstrated the presence of soluble Fe(III)
complexes in aquatic surface sediments (Huettel et al. 1998;
Ratering and Schnell 2000; Taillefert et al. 2000; Nevin and
Lovley 2002). Our findings suggest that FeOB activity could be
partly responsible for the generation of such compounds, whose
presence could signal the occurrence of rapid microscale bacte-
rial Fe redox cycling phenomena.
CONCLUSIONS
Recent studies indicate that although chemolithotrophic
FeOB do not accelerate the oxidation of Fe(II) at circumneutral
pH in a manner analogous to how S-oxidizing bacteria such as
Beggiatoa accelerate dissolved sulfide oxidation at the aerobic-
anaerobic interface (Jorgensen and Revsbech 1983; Nelson et al.
1986), they can nevertheless dominate Fe(II) oxidation in
diffusion-limited opposing gradient systems. Since opposing
gradients of O2and Fe(II) are ubiquitous in nonsulfidogenic
sedimentary environments (including the rhizosphere of aquatic
plants, where a dynamic, microbially catalyzed Fe redox cycle
is likely to exist; (Roden and Wetzel 1996; Emerson et al. 1999;
Frenzel, Bosse, and Janssen 1999; Weiss et al. 2003), bacterial
catalysis may be a widespread mechanism for Fe(II) oxidation
in nature. In addition, by altering the spatial locus of Fe(III)
oxide deposition in the redox transition zone (e.g. through pro-
duction of Fe(III)-binding ligands), FeOB appear to have the
potential to induce rapid microscale coupling of Fe oxidation
and reduction at aerobic-anaerobic interfaces. Further studies
of the distribution and activity of FeOB in relation to their in
situ chemical microenvironment will be required in order fully
understand the biogeochemical cycling of Fe in redox-stratified
systems, and to predict the influence of Fe redox cycling on other
biogeochemical processes in natural environments.
REFERENCES
Arnold RG, Hoffmann MR, DiChristina TJ, Picardal FW. 1990. Regulation
of dissimilatory Fe(III) reduction activity in Shewanella putrefaciens. Appl
Environ Microbiol 56:2811–2817.
Benz M, Brune A, Schink B. 1998. Anaerobic and aerobic oxidation of fer-
rous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria. Arch
Microbiol 169:159–165.
Blake R, Johnson DB. 2000. Phylogenetic and biochemical diversity among
acidophilic bacteria that respire on iron. In: Lovley DR, editor, Environmental
Metal-Microbe Interactions. Washington, DC: ASM Press. pp. 53–78.
390 E. E. RODEN ET AL.
Brendel PJ, Luther GW. 1995. Development of a gold amalgam voltammetric
microelectrode for the determination of dissolved Fe, Mn, O-2, and S(-II) in
porewaters of marine and freshwater sediments. Environ Sci Technol 29:751–
761.
Brock TD, Gustafson J. 1972. Sulfolobus: a new genus of sulfur-oxidizing
bacteria living at low pH and high temperature. Arch Microbiol 84:54–
68.
Caccavo F, Blakemore RP, Lovley DR. 1992. A hydrogen-oxidizing, Fe(III)-
reducing microorganism from the Great Bay estuary, New Hampshire. Appl
Environ Microbiol 58:3211–3216.
Christensen TH, Kjeldsen P, Albrechtsen HJ, Heron G, Nielsen PH, Bjerg PL,
and Holm PE. 1994. Attenuation of landfill leachate polluntants in aquifers.
Crit Rev Environ Sci Technol 24:119–202.
Cornell RM, Schwertmann U. 1996. The Iron Oxides. VCH, New York.
Davison W. 1993. Iron and manganese in lakes. Earth Sci Rev 34:119–163.
Davison W, Seed G. 1983. The kinetics of the oxidation of ferrous iron in
synthetic and natural waters. Geochim Cosmochim Acta 47:67–79.
DiChristina TJ, Delong EF. 1993. Design and application of rRNA-targeted
oligonucleotide probes for the dissimilatory iron- and manganese-reducing
bacterium Shewanella putrefaciens. Appl Environ Microbiol 59:4152–
4160.
Edwards KJ, Bond PL, Gihring TM, Banfield JF. 2000. An archaeal iron-
oxidizing extreme acidophile important in acid mine drainage. Science
287:1701–1876.
Edwards KJ, Rogers DR, Wirsen CO, McCollom TM. 2003. Isolation and char-
acterization of novel psychrophilic, neutrophilic, Fe-oxidizing chemolithoau-
totrophic α- and γ-Proteobacteria from the deep sea. Appl EnvironMicrobiol
69:2906–2913.
Ehrenreich A, Widdel F. 1994. Anaerobic oxidation of ferrous iron by purple
bacteria, a new type of phototrophic metabolism. Appl Environ Microbiol
60:4517–4526.
Ehrlich HL. 1995. Geomicrobiology. New York: Marcel Dekker.
Emerson D. 2000. Microbial oxidation of Fe(II) and Mn(II) at circumneu-
tral pH. In: Lovley DR, editor. Environmental Metal-Microbe Interactions,
Washington, DC: ASM Press. pp. 31–52.
Emerson D, Moyer C. 1997. Isolation and characterization of novel lithotrophic
iron-oxidizing bacteria that grow at circumneutral pH. Appl Environ Micro-
biol 63:4784–4792.
Emerson D, Moyer C. 2002. Neutrophilic Fe-oxidizing bacteria are abundant
at the Loihi Seamount hydrothermal vents and play a major role in Fe oxide
deposition. Appl Environ Microbiol 68:3085–3093.
Emerson D, Revsbech NP. 1994a. Investigation of an iron-oxidizing microbial
mat community located near Aarhus, Denmark: Field studies. Appl Environ
Microbiol 60:4022–4031.
Emerson D, Revsbech NP. 1994b. Investigation of an iron-oxidizing microbial
mat community located near Aarhus, Denmark: Laboratory studies. Appl
Environ Microbiol 60:4032–4038.
Emerson D, WeissJV,Megonigal JP. 1999. Iron-oxidizing bacteria are associated
with ferric hydroxide precipitates (Fe-plaque) on the roots of wetland plants.
Appl Environ Microbiol 65:2758–2761.
Fredrickson JK, Zachara JM, Kennedy DW, Dong H, Onstott TC, Hinman NW,
Li S. 1998. Biogenic iron mineralization accompanying the dissimilatory
reduction of hydrous ferric oxide by a groundwater bacterium. Geochim Cos-
mochim Acta 62:3239–3257.
Frenzel P, Bosse U, Janssen PH. 1999. Rice roots and methanogenesis in a paddy
soil: ferric iron as an alternative electron acceptor in the rooted soil. Soil Biol
Biochem 31:421–430.
Ghiorse WC. 1984. Biology of iron- and manganese-depositing bacteria. Annu
Rev Microbiol 38:515–550.
Harvey PI, Crundwell FK. 1997. Growth of Thiobacillus ferrooxidans:anovel
experimental design for batch growth and bacterial leaching studies. Appl
Environ Microbiol 63:2586–2592.
Huettel M, Ziebis W, Forster S, Luther GW. 1998. Advective transport affecting
metal and nutrient distributions and interfacial fluxes in permeable sediments.
Geochim Cosmochim Acta 62:613–631.
Hunter KS, Wang Y, VanCappellen P. 1998. Kinetic modeling of microbially-
driven redox chemistry of subsurface environments: coupling transport, mi-
crobial metabolism and geochemistry. J Hydrol 209:53–80.
Jahnke R. 1985. A model for microenvironments in deep-sea sediments, forma-
tion and effects on porewater profiles. Limnol Oceanogr 30:956–965.
Johnson DB. 1998. Biodiversity and ecology of acidophilic microorganisms.
FEMS Microbiol Ecol 27:307–317.
Johnson, DB, McGinness S, Ghauri MA. 1993. Biogeochemical cycling of iron
and sulfur in leaching environments. FEMS Microbiol Rev 11:63–70.
Jones JG. 1983. A note on the isolation and enumeration of bacteria which
deposit and reduce ferric iron. J Appl Bact 54:305–310.
Jorgensen BB. 1977. Bacterial sulfate reduction within reduced microniches of
oxidized marine sediments. Mar Biol 41:7–17.
Jorgensen BB. 1989. Biogeochemistry of chemoautotrophic bacteria. In:
Schlegel HG, Bowien B, editors. Biochemistry of Autotrophic Bacteria, Madi-
son, WI: Science Tech Publishers. pp. 117–146.
Jorgensen BB, Revsbech NP. 1983. Colorless sulfur bacteria, Beggiatoa spp.
and Thiovulum spp., in O2and H2S microgradients. Appl Environ Microbiol
45:1261–1270.
Kucera S, Wolfe RS. 1957. A selective enrichment method for Gallionella fer-
ruginea.JBacteriol 74:344–349.
Kusel K, Dorsch T.2000. Effect of supplemental electron donors on the microbial
reduction of Fe(III), sulfate, and CO2in coal mining-impacted freshwater lake
sediments. Microb Ecol 40:238–249.
Kusel K, Dorsch T, Acker G, Stackebrandt E. 1999. Microbial reduction of
Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable
of coupling the reduction of Fe(III) to the oxidation of glucose. Appl Environ
Microbiol 65:3633–3640.
Lovley DR. 1991. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol Rev
55:259–287.
Lovley DR. 2000. Fe(III) and Mn(IV) reduction. In: Lovley DR, editors.
Environmental Metal-Microbe Interactions, Washington, DC: ASM Press.
pp. 3–30.
Lovley DR. 2002. Fe(III)- and Mn(IV)-reducing prokaryotes. In: Dworkin SFM,
Rosenberg E, Schleifer KH, Stackebrandt E. editors. The Prokaryotes,In
press. New York: Springer-Verlag.
Lovley DR, Anderson RT. 2000. The influence of dissimilatory metal reduction
on the fate of organic and metal contaminants in the subsurface. J Hydrol
8:77–88.
Lovley DR, Phillips EJP. 1986. Organic matter mineralization with reduc-
tion of ferric iron in anaerobic sediments. Appl Environ Microbiol 51:683–
689.
Lovley DR, Phillips EJP. 1994. Novel processes for anaerobic sulfate production
from elemental sulfur by sulfate-reducing bacteria. Appl Environ Microbiol
60:2394–2399.
Luther GW, Reimers CE, Nuzzio DB, Lovalvo D. 1999. In situ deployment of
voltammetric, potentiometric and amperometric microelectrodes from a ROV
to determine O2, Mn, Fe, S(−2) and pH in porewaters. Environ Sci Technol
33:4352–4356.
Luther GWI, Kostka JE, Church TM, Sulzberger B, Stumm W. 1992. Seasonal
iron cycling in the salt-marsh sedimentary environment: the importance of
ligand complexes with Fe(II) and Fe(III) in the dissolution of Fe(III) minerals
and pyrite, respectively. Mar Chem 40:81–103.
McCarty PL 1971. Energetics and bacterial growth. In: Faust SD, Hunter JV,
editors. Organic Compounds in Aquatic Environments, New York: Marcel
Dekker, pp. 91–113.
Millero, FJ, Sotolongo S, Izaguirre M. 1987. The oxidation kinetics of Fe(II) in
seawater. Geochim Cosmochim Acta 51:793–801.
Nelson DC, Jorgensen BB, Revsbech NP. 1986. Growth pattern and yield of
a chemoautotrophic Beggiatoa sp. in oxygen-sulfide microgradients. Appl
Environ Microbiol 52:225–233.
Neubauer SC, Emerson D, Megonigal JP. 2002. Life at the energetic edge:
kinetics of circumneutral iron oxidation by lithotrophic iron-oxidizing bac-
teria isolated from the wetland-plant rhizosphere. Appl Environ Microbiol
68:3988–3995.
POTENTIAL FOR MICROSCALE BACTERIAL Fe REDOX CYCLING 391
Nevin KP, Lovley DR. 2002. Mechanisms of Fe(III) oxide reduction in sedi-
mentary environments. Geomicrobiol J 19:141–159.
Peine A, Tritschler A, Kusel K, Peiffer S. 2000. Electron flow in an iron-rich
acidic sediment—evidence for an acidity-driven iron cycle. Limnol Oceanogr
45:1077–1087.
Pronk JT, Johnson DB. 1992. Oxidation and reduction of iron by acidophilic
bacteria. Geomicrobiol J 10:153–171.
Ratering S, Schnell S. 2000. Localization of iron-reducing activity in paddy soil
by profile studies. Biogeochemistry 48:341–365.
Rittmann BE, McCarty PL. 2001. Environmental Biotechnology. Boston:
McGraw-Hill. 754 p.
Roden EE, Wetzel RG. 1996. Organic carbon oxidation and suppression of
methane production by microbial Fe(III) oxide reduction in vegetated and
unvegetated freshwater wetland sediments. Limnol Oceanogr 41:1733–1748.
Schippers A, Jorgensen BB. 2002. Biogeochemistry of pyrite and iron sulfide
oxidation in marine sediments. Geochim Cosmochim Acta 66:85–92.
Silverman MP, Lundgren DG. 1959. Studies on the chemoautotrophic iron
bacterium Ferrobacillus ferrooxidans. II. Manometric studies. J Bacteriol
78:326–331.
Singer PC, Stumm W. 1972. Acid mine drainage–the rate limiting step. Science
167:1121–1123.
Sobolev D, Roden EE. 2001. Suboxic deposition of ferric iron by bacteria in
opposing gradients of Fe(II) and oxygen at circumneutral pH. Appl Environ
Microbiol 67:1328–1334.
Sobolev D, Roden EE. 2002. Evidence for rapid microscale bacterial redox
cycling of iron in circumneutral environments. Anton van Leeuw I81:587–
597.
Sobolev D, Roden E. 2004. Characterization of a neutrophilic, chemolithoau-
totrophic Fe(II)-oxidizing β-Proteobacterium from freshwater wetland sedi-
ments. Geomicrobiol J 21:1–10.
Straub KL, Buchholz-Cleven BEE. 1998. Enumeration and detection of anaer-
obic ferrous-iron oxidizing, nitrate-reducing bacteria from diverse European
sediments. Appl Environ Microbiol 64:4846–4856.
Straub KL, Benz M, Schink B. 2001. Iron metabolism in anoxic environments
at near neutral pH. FEMS Microbiol Ecol 34:181–186.
Straub KL, Benz M, Schink B, Widdel F. 1996. Anaerobic, nitrate-dependent
microbial oxidation of ferrous iron. Appl Environ Microbiol 62:1458–1460.
Stumm W. 1992. Chemistry of the solid-water interface. New York: John Wiley
& Sons.
Stumm W, Morgan JJ. 1996. Aquatic Chemistry. New York: John Wiley & Sons.
Stumm W, Sulzberger B. 1992. The cycling of iron in natural environments:
Considerations based on laboratory studies of heterogeneous redox processes.
Geochim Cosmochim Acta 56:3233–3257.
Taillefert M, Bono A, Luther G. 2000. Reactivity of freshly formed Fe(III)
in synthetic solutions and (pore)waters: voltammetric evidence of an aging
process. Environ Sci Technol 34:2169–2177.
Tebo BM, He LM. 1999. Microbially mediated oxidation precipitation reac-
tions. In: Sparks DL, Grundl TJ, editors. Mineral-Water Interfacial Reactions,
Washington, DC: Americal Chemical Society. pp. 393–414.
Thamdrup B. 2000. Bacterial manganese and iron reduction in aquatic sedi-
ments. Adv Microb Ecol 16:41–84.
Thamdrup B, Fossing H, Jorgensen BB. 1994. Manganese, iron, and sulfur
cycling in a coastal marine sediment, Aarhus Bay, Denmark Geochim Cos-
mochim Acta 58:5115–5129.
Thamdrup B, Finster K, Hansen JW, Bak F. 1993. Bacterial disproportionation
of elemental sulfur coupled to chemical reduction of iron or manganese. Appl
Environ Microbiol 59:101–108.
Thauer RK, Jungermann K, Decker K. 1977. Energy conservation in
chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180.
vandenEnde FP, Meier J, vanGermerden H. 1997. Syntrophic growth of sulfate-
reducing bacteria and colorless sulfur bacteria during oxygen limitation.
FEMS Microbiol Ecol 23:65–80.
vonGunten U, Schneider W. 1991. Primary products of the oxygenation of
iron(II) at an oxic anoxic boundary–nucleation, aggregation, and aging. J
Colloid Interface Sci 145:127–139.
Weiss JV, Emerson D, Backer SM, Megonigal JP. 2003. Enumeration of Fe(II)-
oxidizing and Fe(III)-reducing bacteria in the root zone of wetland plants:
Implications for a rhizosphere iron cycle. Biogeochemistry 64:77–96.
Westermann P. 1993. Wetland and swamp microbiology. In: Ford TE. editor.
Aquatic Microbiology: an Ecological Approach, Boston: Blackwell Scien-
tific, pp. 295–302.
Widdel F, Schnell S, Heising S, Ehrenreich A, Assmus B, Schink B. 1993.
Ferrous iron oxidation by anoxygenic phototrophic bacteria. Nature 362:834–
835.
