GEOMICROBIOLOGY

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A Novel Shewanella Isolate Enhances Corrosion by Using

Metallic Iron as the Electron Donor with Fumarate as the

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Electron Acceptor
Jo Philips,a Niels Van den Driessche,a Kim De Paepe,a Antonin Prévoteau,a Jeffrey A. Gralnick,b Jan B. A. Arends,a
Korneel Rabaeya

a
Center for Microbial Ecology and Technology, Ghent University, Ghent, Belgium
b Department of Plant and Microbial Biology, University of Minnesota, St. Paul, Minnesota, USA

ABSTRACT The involvement of Shewanella spp. in biocorrosion is often attributed to

their Fe(III)-reducing properties, but they could also affect corrosion by using metallic
iron as an electron donor. Previously, we isolated Shewanella strain 4t3-1-2LB from an
acetogenic community enriched with Fe(0) as the sole electron donor. Here, we investi-
gated its use of Fe(0) as an electron donor with fumarate as an electron acceptor and
explored its corrosion-enhancing mechanism. Without Fe(0), strain 4t3-1-2LB fermented
fumarate to succinate and CO2, as was shown by the reaction stoichiometry and pH.
With Fe(0), strain 4t3-1-2LB completely reduced fumarate to succinate and increased the
Fe(0) corrosion rate (7.0 ⫾ 0.6)-fold in comparison to that of abiotic controls (based on
the succinate-versus-abiotic hydrogen formation rate). Fumarate reduction by strain 4t3-
1-2LB was, at least in part, supported by chemical hydrogen formation on Fe(0). Filter-
sterilized spent medium increased the hydrogen generation rate only 1.5-fold, and thus
extracellular hydrogenase enzymes appear to be insufficient to explain the enhanced
corrosion rate. Electrochemical measurements suggested that strain 4t3-1-2LB did not
excrete dissolved redox mediators. Exchanging the medium and scanning electron mi-
croscopy (SEM) imaging indicated that cells were attached to Fe(0). It is possible that
strain 4t3-1-2LB used a direct mechanism to withdraw electrons from Fe(0) or favored
chemical hydrogen formation on Fe(0) through maintaining low hydrogen concentra-
tions. In coculture with an Acetobacterium strain, strain 4t3-1-2LB did not enhance aceto-
genesis from Fe(0). This work describes a strong corrosion enhancement by a She-
wanella strain through its use of Fe(0) as an electron donor and provides insights into its
corrosion-enhancing mechanism.
IMPORTANCE Shewanella spp. are frequently found on corroded metal structures.
Their role in microbial influenced corrosion has been attributed mainly to their
Fe(III)-reducing properties and, therefore, has been studied with the addition of an Received 4 June 2018 Accepted 21 July 2018
electron donor (lactate). Shewanella spp., however, can also use solid electron do- Accepted manuscript posted online 27 July
2018
nors, such as cathodes and potentially Fe(0). In this work, we show that the electron
Citation Philips J, Van den Driessche N, De
acceptor fumarate supported the use of Fe(0) as the electron donor by Shewanella Paepe K, Prévoteau A, Gralnick JA, Arends JBA,
strain 4t3-1-2LB, which caused a (7.0 ⫾ 0.6)-fold increase of the corrosion rate. The Rabaey K. 2018. A novel Shewanella isolate
corrosion-enhancing mechanism likely involved cell surface-associated components enhances corrosion by using metallic iron as
the electron donor with fumarate as the
in direct contact with the Fe(0) surface or maintenance of low hydrogen levels by electron acceptor. Appl Environ Microbiol
attached cells, thereby favoring chemical hydrogen formation by Fe(0). This work 84:e01154-18. https://doi.org/10.1128/AEM
sheds new light on the role of Shewanella spp. in biocorrosion, while the insights .01154-18.
into the corrosion-enhancing mechanism contribute to the understanding of extra- Editor Shuang-Jiang Liu, Chinese Academy of
Sciences
cellular electron uptake processes.
Copyright © 2018 American Society for
Microbiology. All Rights Reserved.
KEYWORDS Acetobacterium, Shewanella fodinae, biocorrosion, complexation,
Address correspondence to Korneel Rabaey,
extracellular electron transfer mechanisms, fumarate fermentation, malate, Korneel.rabaey@ugent.be.
microbially influenced corrosion, solid electron donors, zero-valent iron

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Philips et al. Applied and Environmental Microbiology

C orrosion of iron-based structures entails major costs worldwide, of which about

20% are related to microbially influenced corrosion (MIC) (1). Microorganisms can
affect corrosion through various mechanisms, including metabolizing corrosion prod-
ucts or the metal itself (2–4). Under anoxic conditions, corrosion of metallic iron [Fe(0)]
leads to the formation of hydrogen: Fe(0) ⫹ 2H⫹ ¡ H2 ⫹ Fe2⫹ (⌬G°= ⫽ ⫺10.6 kJ ·
mol⫺1) (reaction A). Initially, it was assumed that microorganisms induce corrosion by
consuming hydrogen, rendering reaction A thermodynamically more favorable (the

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so-called cathodic depolarization theory) (5). Recently, it has become clear that some
strains must have a more efficient mechanism to withdraw electrons from Fe(0), as they
strongly induced corrosion while related hydrogen-consuming strains did not (6–9).
Both the sulfate-reducing strain IS4 (Desulfopila corrodens) and the Methanobacterium
strain IM1 likely use metallic iron directly as an electron donor by means of an
extracellular electron transfer (EET) mechanism (2, 10–12). In contrast, the methanogen
Methanococcus maripaludis releases extracellular enzymes, such as hydrogenases,
which adsorb to the electroactive surface and catalyze reaction A (13). While these
efficient EET mechanisms are undesired in the context of iron corrosion, they are of
interest for the development of biotechnological applications. Microbial electrosynthe-
sis, for instance, powers the microbial production of valuable chemicals from CO2 with
a cathode, which is a solid electron donor like Fe(0) (14, 15). Deutzmann and Spormann
(16) showed that microbial electrosynthesis of acetate and methane could be improved
by using the corrosion-enhancing strain IS4 in coculture with an acetogen or a
methanogen, respectively. So far, the number of iron-corroding microorganisms known
to efficiently withdraw electrons from Fe(0) is limited (3), while a better understanding
of their electron uptake mechanisms is required to assess MIC and develop biotech-
nological applications.
Shewanella spp. are environmentally ubiquitous (17) and are well known for their
ability to use solid electron acceptors, including Fe(III) oxides and anodes (18). Their EET
mechanism involves the Mtr pathway, i.e., an electron conduit that transports electrons
across the outer membrane by a series of membrane-associated c-type cytochromes
(18–20). The Mtr pathway allows Shewanella species to directly interact with solid
electron acceptors, while they also excrete flavins acting as dissolved electron shuttles
(21). Shewanella spp. can also use solid electron donors, such as cathodes, with oxygen
or fumarate as an electron acceptor (22–24). Investigations using the model strain
Shewanella oneidensis MR-1 showed that this strain directly withdraws electrons from a
cathode by reversing the electron flow of the Mtr pathway (23), possibly by redox
bifurcation of the involved flavins (25). So far, only cell maintenance, but not growth,
could be associated with the cathodic electron uptake by strain MR-1 (26).
Shewanella spp. also play a role in MIC, as they are often found on corroded
structures (27–30). Their involvement in MIC has been attributed mostly to their
Fe(III)-reducing properties, which can either enhance or inhibit corrosion (31, 32). Fe(III)
reduction can remove the protective Fe(III) oxide layer on steel (33), while the released
Fe(II) could scavenge oxygen and diminish corrosion (34). In addition, several studies
showed that Shewanella spp. can consume the abiotically generated hydrogen on Fe(0)
(30, 35, 36). As Shewanella spp. can use a cathode as electron donor (22–24), it seems
likely that they can also use other solid electron donors, such as Fe(0). Only recently, it
was suggested that strain MR-1 used carbon steel as an electron donor under nitrate-
reducing conditions (37). Until now, however, the mechanisms used by Shewanella spp.
to withdraw electrons from Fe(0) have not been investigated, and the importance of
this process to MIC remains unclear.
Previously, we isolated a new Shewanella strain from an acetogenic community
enriched with metallic iron as the sole electron donor (J. Philips, E. Monballyu, S. Georg,
K. De Paepe, A. Prévoteau, K. Rabaey, and J. B. A. Arends, submitted for publication).
This Shewanella strain, 4t3-1-2LB, which is highly similar to Shewanella fodinae (Fig. 1), did
not metabolize Fe(0) with CO2 and water as the only possible electron acceptors. For that
reason, the use of metallic iron as an electron donor by strain 4t3-1-2LB was investigated
in this work with the addition of fumarate as an electron acceptor (see Fig. S1A in the

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FIG 1 Maximum-likelihood phylogenetic tree showing the relatedness of Shewanella strain 4t3-1-2LB to representative 16S rRNA gene sequences of all
Shewanella species currently classified in the RDP, Silva, and NCBI databases. The 16S rRNA gene was PCR amplified using primers 63F and 1378R and the
tree was constructed as described elsewhere (Philips et al., submitted).

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supplemental material). The mechanism used by strain 4t3-1-2LB to enhance corrosion was
explored with a series of bottle experiments, as well as electrochemical measurements and
scanning electron microscopy (SEM) (Fig. S1B). In addition, the role of strain 4t3-1-2LB in the
acetogenic enrichment was investigated by testing cocultures of strain 4t3-1-2LB and an
acetogenic isolate (Fig. S1C). This study describes a strong increase of the rate of corrosion
by a Shewanella strain through its use of Fe(0) as the electron donor when a suitable
electron acceptor is present.

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RESULTS
Shewanella strain 4t3-1-2LB converts fumarate in the presence and absence of
Fe(0). The first experiment evaluated the use of Fe(0) as the electron donor by strain
4t3-1-2LB with fumarate as the electron acceptor (see Fig. S1A in the supplemental
material). In abiotic controls with Fe(0), the fumarate concentration remained constant,
while no other volatile fatty acids (VFAs) were formed (Fig. 2A). Very recently, Fe(0) was
reported to abiotically reduce CO2 to acetate and other VFAs at high temperatures and
CO2 pressures (38), but the conditions in our experiments likely did not favor such
reactions. In our abiotic controls, the hydrogen concentration increased linearly over
time with a rate (0.49 ⫾ 0.04 mmol of electron equivalents [meeq] · liter⫺1 · day⫺1 over
14 days) (Fig. 2A) that was highly similar to that observed in our other study (0.48 ⫾
0.05 meeq liter⫺1 · day⫺1) (Philips et al., submitted). Dissolved Fe(II) concentrations
remained low and did not increase concomitantly with the hydrogen concentration,
likely because part of the Fe(II) precipitated as Fe(II) carbonates or oxyhydroxides
(whitish precipitates were visible). In the abiotic controls, the iron powder remained
loose (there was visual turbidity upon shaking) throughout the experiment (see Fig. S2
in the supplemental material).
In the presence of Fe(0), strain 4t3-1-2LB immediately started to reduce fumarate to
succinate, while malate was transiently formed (Fig. 2B). As long as fumarate or malate
was present (until day 11), succinate concentrations increased quasilinearly with an
average rate of 3.43 ⫾ 0.10 meeq · liter⫺1 · day⫺1. Final succinate concentrations were
equal to the initial fumarate concentration (20 mM). Hydrogen concentrations were
below the detection limit (⬍0.06 meeq liter⫺1 or 0.04% [vol/vol] in the headspace) as
long as succinate was being produced, but they started to increase after fumarate
depletion (day 11). These findings show that fumarate reduction was at least in part
supported by hydrogen consumption. Dissolved Fe(II) concentrations initially increased
to a level higher than that in the abiotic controls, but they decreased after day 6 (Fig.
2B), likely due to precipitation of Fe(II) salts, just as in the abiotic controls. During
fumarate reduction, the liquid phase was colored slightly yellow (Fig. S2), possibly
because of Fe(III) formation. Dissolved Fe(III) concentrations in this study were always
low in comparison to Fe(II) concentrations, which hindered their accurate measurement
by the used method (39). Upon fumarate depletion, the liquid phase turned colorless
again and became clear (with no turbidity upon shaking), because all iron powder
aggregated at the bottom of the bottle (Fig. S2). A second addition of fumarate (20 mM)
led to similar concentration changes (see Fig. S3 in the supplemental material). The
yellow color of the liquid phase appeared again during fumarate reduction and
disappeared after fumarate depletion. The iron powder remained aggregated at the
bottom of the bottle and became overlaid with a dark green layer of precipitates (Fig. S2).
Strikingly, in the absence of Fe(0), strain 4t3-1-2LB also converted fumarate to
succinate (Fig. 2C). Malate was again formed transiently, but succinate production
started only after an initial lag phase, and the final succinate concentrations were
20.5% ⫾ 4.4% lower than the initial fumarate concentrations (day 14) [final succi-
nate concentrations in the absence of Fe(0) were significant lower than those in the
presence of Fe(0), based on a Student t test [P ⬍ 0.01]). Fumarate conversion
continued after a second fumarate addition (Fig. S3), demonstrating that the
reduction of fumarate was not just supported by the limited amount of yeast
extract present in the medium [0.1 g · liter⫺1, which corresponds to 18 meeq ·
liter⫺1, assuming that yeast extract has the typical chemical composition of biomass

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FIG 2 Change of fumarate, succinate, malate, hydrogen, and dissolved Fe(II) concentrations, expressed
as 10⫺3 electron equivalents per volume of the liquid phase (meeq · liter⫺1), over time for abiotic controls
with fumarate (A), Shewanella strain 4t3-1-2LB in the presence of Fe(0) powder and fumarate (B), and
Shewanella strain 4t3-1-2LB with fumarate but in the absence of Fe(0) (C). Plotted values are averages
from triplicate bottles, and error bars show the standard deviation.

(C5H7O2N) and is completely oxidized (40)]. In the absence of Fe(0), Shewanella

visibly grew and remained planktonic (with an optical density increase from 0.01 to
a maximum of 0.16).
Fumarate conversion by strain 4t3-1-2LB in the presence and absence Fe(0) was
further analyzed by determining the CO2 content in the headspace and the pH at the
end of the experiment (day 25, after two conversions of 20 mM fumarate) (Fig. 3). In
abiotic controls without Fe(0), the CO2 content remained similar to the initial level (10%)
(Fig. 3A). In abiotic controls with Fe(0), the CO2 content had decreased to 4.7% ⫾ 0.4% over
25 days, likely because CO2 was partly removed from the headspace by Fe(II) carbonate
formation. Fumarate conversion by strain 4t3-1-2LB in the presence of Fe(0) led to a
much lower CO2 level (final headspace content, 0.4% ⫾ 0.1%) than in the abiotic
controls, suggesting that more Fe(II) carbonate precipitates had been formed or
possibly that CO2 had been consumed by a biological process (S. fodinae can grow with

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FIG 3 Headspace CO2 content (A) and pH in the liquid phase (B) at the end of the experiment (day 25)
for the different treatments shown in Fig. 2 and S3 as well as an abiotic control without Fe(0). Plotted
values are averages from triplicate bottles, and error bars show the standard deviation.

bicarbonate as a carbon source [41]). In contrast, in the absence of Fe(0), fumarate

conversion by strain 4t3-1-2LB increased the CO2 content (final headspace content,
14.4% ⫾ 0.3%) in comparison to the initial level, demonstrating the production of CO2
(an additional 0.20 ⫾ 0.01 mmol CO2 in the headspace). Furthermore, there were clear
differences in the pH between the different treatments (Fig. 3B). In abiotic controls
without Fe(0), the pH remained close to the initial value of 7.20 [the pKa of 3-(N-
morpholino)propanesulfonic acid (MOPS)], while in abiotic controls with Fe(0), the pH
slightly increased (final pH, 7.30 ⫾ 0.01). Fumarate conversion by strain 4t3-1-2LB with
Fe(0) led to a significant increase of the pH to 7.91 ⫾ 0.06 (Student t test, P ⬍ 0.01),
while without Fe(0), the pH (7.13 ⫾ 0.02) had slightly decreased in comparison to the
initial value.
These differences in stoichiometry, pH, and CO2 content show that strain 4t3-1-2LB
converted fumarate differently with and without Fe(0). As discussed in detail below,
fermentation of fumarate to succinate and CO2 most likely occurred in the absence of
Fe(0) (with fumarate as an electron acceptor and donor), while the complete reduction
of fumarate to succinate occurred in the presence of Fe(0). This shows that strain
4t3-1-2LB could use Fe(0) as the electron donor. Furthermore, the rate of succinate
formation by strain 4t3-1-2LB with Fe(0) as an electron donor was (7.0 ⫾ 0.6)-fold
higher than the hydrogen formation rate under abiotic conditions (Fig. 2), demonstrat-
ing that strain 4t3-1-2LB significantly increased the Fe(0) corrosion rate in comparison
to abiotic conditions (significant difference based on a Student t test [P ⬍ 0.001]). The
real corrosion enhancement factor might even have been higher than 7.0 ⫾ 0.6, since
the higher pH at the end of the fumarate conversion by strain 4t3-1-2LB with Fe(0) (Fig.
3) implies a lower chemical hydrogen formation rate (reaction A).
The use of Fe(0) as an electron donor by strain 4t3-1-2LB was also evaluated with
other electron acceptors, including sodium nitrate, trimethylamine oxide (TMAO), and
dimethyl sulfoxide (DMSO) (all at 20 mM). Nitrate and TMAO, however, led to a strong

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FIG 4 Change of malate, succinate, hydrogen, and dissolved Fe(II) concentrations, expressed in 10⫺3
electron equivalents per volume of the liquid phase (meeq · liter⫺1), over time for abiotic controls with
Fe(0) and succinate (A) or malate (B). Plotted values are averages from triplicate bottles, and error bars
show the standard deviation.

chemical oxidation of the iron powder (clear from the extensive brown precipitation)
(42–44), while DMSO did not support the growth of strain 4t3-1-2LB (results not shown).
Malate complexes Fe(II), but this does not explain the enhanced Fe(0) corro-
sion by strain 4t3-1-2LB. Some organic compounds, including malate, are known to
form complexes with ferric and ferrous iron (45, 46). For this reason, abiotic controls
with 20 mM malate or succinate were investigated (Fig. S1A). Abiotic controls with
succinate (Fig. 4A) showed concentration changes very similar to those of the abiotic
controls with fumarate (Fig. 2A). The hydrogen concentration increased with an aver-
age rate of 0.40 ⫾ 0.01 meeq · liter⫺1 · day⫺1 (over 21 days), while dissolved Fe(II)
concentrations remained low. With malate, however, the dissolved Fe(II) concentration
increased with an average rate of 0.34 ⫾ 0.03 meeq · liter⫺1 · day⫺1 (over 21 days),
almost proportionally to the hydrogen formation rate (0.26 ⫾ 0.03 meeq · liter⫺1 ·
day⫺1, over 21 days) (Fig. 4B). The flushing of the headspace at the start of the
experiment, which removed hydrogen but not Fe(II), explains the higher dissolved Fe(II)
concentration at day 0 in comparison to the initial hydrogen concentration (Fig. 4B).
The increasing dissolved Fe(II) concentration with malate suggests that malate was able
to keep all Fe(II) formed in solution by complexation. The chelating properties of malate
were also clear from liquid samples exposed to air. Oxygen in the air oxidized the
dissolved Fe(II) in the samples to Fe(III), which usually resulted in an extensive brown
precipitation, but with malate, liquid samples were only slightly colored brown and
remained completely clear (see Fig. S4 in the supplemental material). In addition,
malate diminished the abiotic hydrogen formation rate (0.26 ⫾ 0.03 meeq · liter⫺1 ·
day⫺1) in comparison to those with fumarate and succinate (Fig. 2A and 4; see Fig. 9)
(significant differences, based on Student t tests with a P value of ⬍0.01). With both
malate and succinate, the pH at the end of the experiments was similar to that with

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FIG 5 Change of fumarate, malate, succinate, hydrogen, and dissolved Fe(II) concentrations, expressed
in 10⫺3 electron equivalents per volume of the liquid phase (meeq · liter⫺1), for the medium exchange
test with Shewanella strain 4t3-1-2LB. At day 12, the liquid phase was replaced by fresh medium (40 ml)
with 20 mM fumarate (A) or without fumarate (B), while the Fe(0) powder was retained in the bottle.
Plotted values are averages from triplicate bottles, and error bars show the standard deviation.

fumarate (results not shown); thus, the difference in hydrogen formation rates is not pH
related. Most likely, the complexing properties of malate also explain its lower hydro-
gen formation rate, which is discussed further below. The formation of malate, there-
fore, is not an explanation for the strongly increased rate of Fe(0) corrosion by strain
4t3-1-2LB with fumarate as the electron acceptor.
Cells attached to Fe(0) are responsible for fumarate reduction. The corrosion-
enhancing mechanism of strain 4t3-1-2LB was first explored by exchanging the me-
dium of strain 4t3-1-2LB growing on Fe(0) and fumarate (Fig. S1B). After the depletion
of 20 mM fumarate (day 12), the liquid phase was replaced with 40 ml fresh anaerobic
defined medium, while the aggregated iron powder remained in the bottle. Bottles
were briefly flushed with N2-CO2 gas to remove any hydrogen. After the medium
exchange, 20 mM fumarate or no electron acceptor was added. With the addition of
fumarate, succinate formation after the medium exchange continued without a lag
phase and with a rate (5.04 ⫾ 0.07 meeq · liter⫺1 · day⫺1 between days 12 and 19) even
higher than that before the medium exchange (4.15 ⫾ 0.06 meeq · liter⫺1 · day⫺1
during the first 7 days) (significantly different based on a Student t test [P ⬍ 0.01]) (Fig.
5A). These findings suggest that attached cells were responsible for the fumarate
reduction, as planktonic cells were removed from the liquid phase. Moreover, the glass
and the liquid phase were always completely clear (Fig. S2), suggesting that the cells
must have been attached to the Fe(0) powder. Accordingly, SEM imaging showed
attachment of cells to the Fe(0) powder (Fig. 6). Rod-shaped cells were seen clustered
together in cavities of the iron powder particles. The Fe(0) surface was largely covered

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FIG 6 SEM image of Shewanella strain 4t3-1-2LB cells clustered together in a cavity of an iron powder
granule after 14 days of growth with fumarate as the electron acceptor. Arrows point to cells (C) or
precipitates (P).

by precipitates of various sizes and shapes. The outside of the Shewanella cells looked
coarse, which possibly indicates that precipitates had also formed on the outer surfaces
of the cells.
The increased electron uptake from Fe(0) by strain 4t3-1-2LB could be mediated by
hydrogen or formate (13). Without the addition of an electron acceptor after the
medium exchange, hydrogen was formed at a rate (0.70 ⫾ 0.10 meeq · liter⫺1 · day⫺1
between days 12 and 22) (Fig. 5B) higher than the abiotic hydrogen formation rate (a
significant difference based on a Student t test [P ⬍ 0.0001]; the pHs were similar) (Fig.
2A), but still much lower than the succinate formation rate after exchanging the
medium and adding fumarate (Fig. 5A). Similar hydrogen formation rates were ob-
tained when the medium was not exchanged after fumarate depletion (results not
shown). Formate concentrations always remained low (⬍1 meeq · liter⫺1) in this study
(results not shown). Consequently, the strong increase of the corrosion rate by strain
4t3-1-2LB cannot be explained solely by a kinetic stimulation of the hydrogen (or
formate) formation reaction on the Fe(0) surface, for instance, by adsorbed extracellular
hydrogenase (or formate dehydrogenase) enzymes.
The results of the medium exchange also suggest that no dissolved, self-excreted
redox mediators were involved in the electron uptake from Fe(0) by strain 4t3-1-2LB.
This was confirmed by electrochemical measurements on filtered liquid samples. Cyclic
voltammograms measured with a glassy carbon rotating-disk electrode (scan rate, 10
mV · s⫺1; rotation speed, 2,000 rpm) were similar for strain 4t3-1-2LB grown with Fe(0)
and fumarate and for an abiotic control with Fe(0); nevertheless, there were strong
differences in the height of peaks, likely related to Fe(II) reduction and oxidation (see
Fig. S5A in the supplemental material). Addition of 10 ␮M riboflavin to the filter liquid
samples, i.e., an exogenous dissolved redox mediator, resulted in an additional sigmoi-
dal feature in the voltammogram, which was otherwise absent (Fig. S5B). This further
confirmed that strain 4t3-1-2LB did not excrete dissolved redox mediators to the liquid
phase at a detectable concentration in our experiments (47).
Defined spent medium slightly increases the hydrogen formation rate. The
corrosion-enhancing mechanism of strain 4t3-1-2LB was further assessed by investi-
gating the effect of filter-sterilized spent medium on the corrosion of Fe(0) (Fig. S1B).
The medium removed in the medium exchange experiment was filter sterilized and
placed in bottles with 5 ml medium and 2 g Fe(0) powder (spent defined medium).
Dissolved Fe(II) concentrations remained low in these bottles (Fig. 7A), while hydrogen
was formed at a rate (0.72 ⫾ 0.03 meeq · liter⫺1 · day⫺1, over 24 days) similar to the
hydrogen formation rate after the medium exchange without fumarate addition (Fig.
5B) and higher than the abiotic hydrogen formation rate (Fig. 2A) (a significant

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FIG 7 Change of fumarate, malate, succinate, hydrogen, and dissolved Fe(II) concentrations, expressed
in 10⫺3 electron equivalents per volume of the liquid phase (meeq · liter⫺1), over time for spent defined
medium (A) and spent LB medium (B) (both filter sterilized) added to fresh Fe(0) powder. Plotted values
are averages from triplicate bottles, and error bars show the standard deviation.

difference based on a Student t test [P ⬍ 0.01]; the pHs were similar). The increase in
the hydrogen formation rate could be due to the catalytic effect of extracellular
hydrogenase enzymes present in the spent medium (13). However, their catalyzing
effect on the hydrogen formation reaction is alone insufficient to explain the large
increase in the rate of corrosion by strain 4t3-1-2LB with fumarate as an electron
acceptor.
In addition, spent Luria-Bertani (LB) medium was tested by adding 1 ml filter-
sterilized supernatant of Shewanella cells aerobically grown in LB (no fumarate addi-
tion) to bottles with 40 ml anaerobic defined medium with fumarate and Fe(0) powder.
The bottles were briefly flushed with N2-CO2 gas to remove traces of oxygen. The
fumarate concentrations in these bottles exponentially decreased, and fumarate
was incompletely converted into only malate (Fig. 7B). No fumarate conversion
occurred in controls to which fresh LB medium was added (results not shown). The
hydrogen formation rate (0.26 ⫾ 0.01 meeq · liter⫺1 · day⫺1, between days 0 and
21) and the linearly increasing dissolved Fe(II) concentrations (Fig. 7B) were similar
to those in abiotic controls with malate (Fig. 4B). These results suggest that the
spent LB medium contained fumarate hydratase enzymes (catalyzing the reversible
hydration of fumarate to malate) (discussed below), most likely resulting from the
lysis of the cells grown in the LB medium, but no hydrogenase enzymes affecting
the Fe(0) corrosion rate.
Shewanella strain 4t3-1-2LB does not increase acetogenesis from Fe(0) by an
Acetobacterium isolate. It was previously found that a corrosion-enhancing strain
(sulfate-reducing strain IS4) improved the electron transfer from a cathode for
acetogenesis (16). In order to investigate whether Shewanella strain 4t3-1-2LB
played a similar role in the acetogenic enrichment from which it was isolated,

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FIG 8 Change of acetate, formate, fumarate, malate, succinate, and hydrogen concentrations, expressed
in 10⫺3 electron equivalents per volume of the liquid phase (meeq · liter⫺1), over time with Acetobac-
terium strain 73-4-6p-4 (A), Shewanella strain 4t3-1-2LB and Acetobacterium strain 73-4-6p-4 inoculated
together at day 0 (B), and Acetobacterium strain 73-4-6p-4 inoculated at day 14 after Shewanella strain
4t3-1-2LB had completed the reduction of 20 mM fumarate (C). Plotted values are averages from
triplicate bottles, and error bars show the standard deviation. Note the different scale of the x axis for
panel C.

acetogenesis from Fe(0) by cocultures of Shewanella strain 4t3-1-2LB and Acetobac-

terium strain 73-4-6p-4 was investigated (Fig. S1C). Acetobacterium strain 73-4-6p-4
by itself produced acetate at an average rate of 0.85 ⫾ 0.19 meeq · liter⫺1 · day⫺1
(over 21 days), while hydrogen concentrations remained below the detection limit
(Fig. 8A). This acetate production rate corresponds to a (1.77 ⫾ 0.43)-fold corrosion
enhancement in comparison to hydrogen formation under abiotic conditions,
which is similar to results from our other study (Philips et al., submitted). Adding
Acetobacterium strain 73-4-6p-4 and Shewanella strain 4t3-1-2LB together from the
start of the experiment resulted in a similar acetate production rate (0.80 ⫾ 0.05 meeq ·
liter⫺1 · day⫺1, over 21 days) (Fig. 8B). Also, when Acetobacterium strain 73-4-6p-4 was
added after strain 4t3-1-2LB had reduced 20 mM fumarate, acetogenesis had a similar rate
(0.83 ⫾ 0.04 meeq · liter⫺1 · day⫺1, between days 14 and 35) (Fig. 8C). These results show
that strain 4t3-1-2LB was not able to enhance acetogenesis from Fe(0) by Acetobacterium
strain 73-4-6p-4.
For comparison, all product formation rates, i.e., approximations for the corrosion
rates, obtained in this study are summarized in Fig. 9.

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DISCUSSION
Shewanella strain 4t3-1-2LB ferments fumarate to succinate and CO2 in the
absence of Fe(0). Shewanella strain 4t3-1-2LB was able to convert fumarate to succi-
nate in the presence and absence of Fe(0) (Fig. 2). Differences in stoichiometry, pH, and
CO2 content suggested that different metabolic processes occurred with and without
Fe(0) (Fig. 2 and 3). In the absence of an electron donor, strain 4t3-1-2LB most likely
performed a fumarate fermentation by coupling the reduction of six moles of fumarate to
succinate with the oxidation of one mole of fumarate to CO2 (ΔG°= values were calculated

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as described by Madigan et al. [48]): 7C4H2O42⫺ ⫹ 8H2O ¡ 6 C4H4O42⫺ ⫹ 4HCO3⫺ ⫹ 2H⫹
(⌬G°= ⫽ ⫺63.09 kJ · mol⫺1 fumarate) (reaction B). In the presence of Fe(0) as the electron
donor, the complete reduction of fumarate to succinate coupled to the oxidation of Fe(0)
is thermodynamically more favorable than fumarate fermentation: C4H2O42⫺ ⫹ Fe(0) ⫹
2H⫹ ¡ C4H4O42⫺ ⫹ Fe2⫹ (⌬G°= ⫽ ⫺85.23 kJ · mol⫺1 fumarate) (reaction C). These reactions
are in good agreement with the pH evolutions observed in our experiments (Fig. 3). In
addition, the measured formation of 0.20 mmol CO2 in the headspaces of bottles of strain
4t3-1-2LB in the absence of Fe(0) corresponds well with the expected increase of 0.34 mmol
based on reaction B, as part of the formed CO2 remained in solution as bicarbonate.
Moreover, the theoretical stoichiometry of 6:7 (86%) for succinate-fumarate is in agreement
with the observed ratio (80%), as part of the fumarate was likely used for biomass
formation.
The same fumarate fermentation reaction (reaction B) was reported for Providencia
rettgeri (49), Malonomonas rubra (50), and Syntrophobacter fumaroxidans (51). Besides
fumarate fermentation, S. fumaroxidans can also couple the complete reduction of
fumarate to succinate with the oxidation of electron donors, such as hydrogen.
Geobacter sulfurreducens converts fumarate to succinate with a 1:0.8 stoichiometric
ratio in the absence of an electron donor, but it is unable to grow with this reaction (52).
In addition, Shewanella amazonensis is able to grow on fumarate in the absence of an
electron donor (J. Gralnick, unpublished results), suggesting that fumarate fermenta-
tion may be common within the genus Shewanella. Consequently, experiments study-
ing microbial conversions with fumarate as an electron acceptor should always carefully
evaluate whether decreasing fumarate concentrations are due to the oxidation of an
electron donor or due to fumarate fermentation.
Transient formation of malate leads to Fe(II) and Fe(III) complexation and dimin-
ishes the abiotic hydrogen formation rate. Malate was transiently formed during the
conversion of fumarate by strain 4t3-1-2LB, independently of the presence of Fe(0) (Fig.
2). Hydration of fumarate to malate is a reversible reaction that does not require energy
and is normally catalyzed by the enzyme fumarate hydratase (fumarase). This enzyme
participates in the tricarboxylic acid (TCA) cycle of many organisms, including She-
wanella spp. (53). This or a similar enzyme was released by strain 4t3-1-2LB during
aerobic growth in LB medium (likely by cell lysis), as the spent LB medium also
converted fumarate to malate (Fig. 7B). Fumarate conversion by G. sulfurreducens also
led to transient formation of malate (52).
Malate is known to form stable complexes with Fe(II) and Fe(III), which prevents
Fe(II) and Fe(III) precipitation at circumneutral pH (45, 46, 54). In our experiment, this
was clear from the increase of the dissolved Fe(II) concentration proportionally to the
hydrogen concentration in abiotic controls with malate (Fig. 4B). Malate was likely
capable of keeping Fe(II) in solution as long as its concentration was higher than the
Fe(II) concentration (46), which explains why dissolved Fe(II) concentrations decreased
after day 2 to 6 with strain 4t3-1-2LB growing on Fe(0) and fumarate (Fig. 2B and 5).
Complexation of Fe(III) by malate could also explain the yellow color of the liquid
phase during fumarate reduction by strain 4t3-1-2LB (see Fig. S2 in the supplemental
material), as Fe(III) is usually not soluble. As described above, dissolved Fe(III) concen-
trations were low in this study and could not be accurately measured. It is not clear if
the formed Fe(III) resulted from the oxidation of Fe(II) by trace amounts of oxygen
intruded into the bottles or possibly by strain 4t3-1-2LB itself. Based on the color of the
crust overlaying the aggregated Fe(0) powder (Fig. S2), green rust formation possibly

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FIG 9 Comparison of product formation rates (meeq · liter⫺1 · day⫺1) with Fe(0) (as described in the text),
which are approximations for the corrosion rate. S., Shewanella strain 4t3-1-2LB; A., Acetobacterium strain
73-4-6p-4. The letters in the bars indicate statistically different groups as determined with a Duncan test
(␣ ⫽ 0.05); corrosion rates increase in alphabetical order.

occurred. This further suggests the presence of Fe(III), as green rust is typically a mixture
of Fe(II) and Fe(III) hydroxides. Interestingly, various Shewanella strains were already
reported to form green rust by reducing Fe(III) oxides (55, 56). Further mineralogical
characterizations will be required to verify if the dark green crust indeed consisted of
green rust.
Malate also diminished the abiotic hydrogen formation rate in comparison to those
with fumarate and succinate (Fig. 4 and 9). Several organic anions, including malate,
were already described as good inhibitors of steel corrosion (57). This is likely due to the
adsorption with their functional groups on the Fe(0) surface, thereby partially blocking
the access for water, which is required for corrosion (58).
Our results show that corrosion rates cannot be correctly assessed from dissolved
Fe(II) concentrations alone [for instance, higher dissolved Fe(II) concentrations but a
lower hydrogen formation rate in abiotic controls with malate versus fumarate (Fig. 4B
and 9)], even though this often occurs in biocorrosion studies. As an alternative to a
destructive extraction of Fe(II) and Fe(III) with acid, the addition of a biologically inert
chelator, such as EDTA, could help with correctly assessing corrosion rates in MIC
studies by keeping Fe(II) and Fe(III) in solution. Nevertheless, the possible effect of such
a chelator on the corrosion process itself needs to be well understood first.
Shewanella strain 4t3-1-2LB possibly uses a direct EET mechanism. Strain 4t3-
1-2LB had an Fe(0) corrosion rate significantly higher than that of any of the abiotic
controls or spent-medium tests (Fig. 9). Hydrogen consumption was at least in part
responsible for fumarate reduction by strain 4t3-1-2LB, as hydrogen concentrations
remained below the detection limit during succinate formation (Fig. 2B). Deutzmann et
al. (13) previously demonstrated that cell-free spent medium of Methanococcus mari-
paludis enhanced the hydrogen and formate formation from Fe(0). In addition, our
upcoming study found that the increased hydrogen generation by filter-sterilized spent
medium could explain the Fe(0) corrosion enhancement by several acetogens (Philips
et al., submitted). The components in the spent medium responsible for this increased
hydrogen generation are likely free extracellular hydrogenase enzymes, which adsorb
on the electroactive surface and catalyze the formation of hydrogen (13, 59, 60). Also
in this study, spent defined medium increased the rate of hydrogen formation from
Fe(0) in comparison to the abiotic hydrogen formation rate (Fig. 9). A similar increased
hydrogen formation rate was measured after exchanging the medium without adding
an electron acceptor. Nevertheless, these rates were much lower than the rate of
corrosion by strain 4t3-1-2LB with fumarate (Fig. 9). Consequently, the corrosion-
enhancing mechanism of strain 4t3-1-2LB cannot be based solely on a kinetic stimu-

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FIG 10 Schematic overview of different possible mechanisms for electron uptake from Fe(0) by strain
4t3-1-2LB, contradicted or suggested by the results of this study.

lation of the chemical hydrogen formation reaction on the Fe(0) surface by extracellular
components (Fig. 10).
Interestingly, spent LB medium led to a decreased hydrogen formation rate, due to
the formation of malate (0.26 ⫾ 0.01 meeq · liter⫺1 · day⫺1) (Fig. 7B and 9). Shewanella
spp. do not express hydrogenases during aerobic growth (LB medium) (61), while
during anaerobic growth on Fe(0), strain 4t3-1-2LB likely expressed hydrogenases to
consume the chemically formed hydrogen. This shows that strong differences in the
presence and concentration of extracellular enzymes can arise from different types of
spent medium and different growth conditions, which will be of major importance for
further studies investigating microorganisms whose EET mechanism relies on extracel-
lular enzymes.
Exchanging the medium (Fig. 5A), as well as electrochemical measurements (see Fig.
S5 in the supplemental material), showed that the corrosion-enhancing mechanism of
strain 4t3-1-2LB was also not based on the excretion of dissolved redox mediators (Fig.
10). In contrast, exchanging the medium of S. oneidensis MR-1 grown on an anode
strongly diminished its current production (62), demonstrating the importance of
electron shuttling by its excreted flavins (21). Flavins possibly also played a role in
catalyzing cathodic oxygen reduction by Shewanella loihica (24), while Shewanella
putrefaciens likely did not use a redox mediator to reduce oxygen with cathodic
electrons (22).
Both the medium exchange experiment and SEM imaging showed that cells were
attached to the Fe(0) powder (Fig. 5 and 6). For this reason, it is possible that strain
4t3-1-2LB used a direct mechanism to obtain electrons from Fe(0) (Fig. 10). This could
entail a reversal of the Mtr pathway, similarly as was described for S. oneidensis MR-1
reducing fumarate or oxygen with a cathode as an electron donor (23, 25, 26). Those
studies, however, first grew strain MR-1 on an anode, after which the electrode
potential was lowered and fumarate or oxygen was added, followed by the examina-
tion of the mechanism within hours. This experimental procedure could have strongly
affected the EET mechanism used by strain MR-1. Moreover, the reversal of the Mtr
pathway was recently found not to support cell growth but to support only cell
maintenance (26). In our experiments, strain 4t3-1-2LB grew with Fe(0) as an electron
donor (not experimentally measured), as it would otherwise have favored fumarate
fermentation. Further investigations, for instance, using mutant strains and electro-
chemical characterizations of cells grown on cathodes, will be required to assess
whether a reversal of the Mtr pathway could be involved in the uptake of extracellular
electrons by strain 4t3-1-2LB.
Finally, another plausible explanation for the corrosion enhancement by strain
4t3-1-2LB could be that attached cells scavenge hydrogen on the Fe(0) surface and
thereby cause a thermodynamic shift of reaction A (Fig. 10). This cathodic depolariza-

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Metallic Iron as an Electron Donor for Shewanella Applied and Environmental Microbiology

tion theory has in recent literature been contested by the finding that only strains
isolated with Fe(0) were found to enhance corrosion, while typical hydrogen-
consuming strains did not (2, 6, 8, 9). Those studies, however, did not evaluate
differences in the hydrogen threshold (minimum hydrogen level allowing microbial
growth) between strains, even though it seems likely that enrichments with Fe(0) lead
to strains better adapted to use low hydrogen concentrations than strains enriched
with a hydrogen overpressure. Fumarate reducers typically have one- and two-orders-

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of-magnitude-lower hydrogen thresholds than methanogens and acetogens, respec-
tively (63). Possibly this explains the much higher corrosion enhancement factor
obtained in this study in comparison to that of acetogens (Fig. 8A). Further investiga-
tions, for instance, testing mutant strains incapable of hydrogen consumption (64), will
be required to investigate the involvement of hydrogen in the electron uptake mech-
anism of strain 4t3-1-2LB.
Ecological relevance and technological implications. The genus Shewanella had
only a low abundance (0.6%) in the acetogenic enrichment from which Shewanella
strain 4t3-1-2LB was isolated (Philips et al., submitted). Nevertheless, Shewanella cells
must have been active in the enrichment, as they survived five transfers of the
enrichment and grew quickly once transferred to LB medium. Coculturing Shewanella
strain 4t3-1-2LB with an Acetobacterium isolate did not increase acetogenesis from Fe(0)
by the Acetobacterium strain (Fig. 8). In contrast, the corroding IS4 strain increased
acetate and methane production in coculture with Acetobacterium woodii and M.
maripaludis, respectively, on a cathode (16). Also, the composition of microbial com-
munities on acetogenic cathodes has already led to the suggestion that extracellular
electrons could be withdrawn by electrotrophs, such as Desulfovibrio spp., which pass
them as hydrogen to acetogens (65). Our results show that strain 4t3-1-2LB definitely
did not play such a syntrophic role. This is likely because it does not increase the
formation of available hydrogen from Fe(0) (Fig. 5B), in contrast to, for instance, strain
IS4 (6, 16). Consequently, the function of strain 4t3-1-2LB in the acetogenic community
remains unclear. It is possible that it survived by consuming metabolites released by the
acetogenic community, such as fumarate, possibly in mixotrophy with Fe(0) as the
electron donor.
Shewanella spp. and related species are likely important for MIC, as they were
present in biofilms on corroded steel (27, 66), dominated the microbial community of
water exposed to metal materials (29), and have frequently been isolated from envi-
ronments with corroded structures (28, 30, 67), as in our previous work (Philips et al.,
submitted). Until now, the importance of Shewanella spp. for MIC was mainly related to
their Fe(III)-reducing properties. For this reason, their effect on corrosion was usually
studied by the addition of a suitable electron donor, i.e., lactate (31, 33–35, 66). Only a
few studies examined the effect of Shewanella spp. on corrosion with the addition of
electron acceptors, such as sulfite or ferric citrate (35) or nitrate (36, 37). Only small
corrosion enhancements were found (1.3-fold with nitrate [measured after 20 h] [36] to
4.2-fold with sulfite [but with lactate present] [35]), which were indirectly linked to the
metabolism of the Shewanella strains. Only recently, nitrate was reported to support
Fe(0) oxidation by strain MR-1 (corrosion enhancement factor without lactate, about
1.3), but it remained unclear to what level corrosion was caused by the bacterial
metabolism or by the corrosive effect of the produced nitrite (37). In contrast, our study
found a strong ([7.0 ⫾ 0.6]-fold) increase of the corrosion rate by a Shewanella strain
solely by the use of Fe(0) as an electron donor in the presence of an electron acceptor
(here fumarate). Fumarate likely occurs only in low concentrations in the environment,
but Shewanella spp. can use various other electron acceptors, including environmen-
tally ubiquitous oxygen, nitrate, or Fe(III). The effect of these electron acceptors on MIC
by Shewanella spp. should be reevaluated, as our study suggests that Shewanella spp.
could strongly aggravate corrosion. Future studies should also use more-realistic
iron-based materials (for example steel coupons), instead of Fe(0) powder as used in

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Philips et al. Applied and Environmental Microbiology

this study, to correctly assess the possibly large contribution of Shewanella spp. to
biocorrosion.
Besides its undesirable induction of corrosion, strain 4t3-1-2LB could be of interest
for biotechnological applications, such as microbial conversions powered by cathodes.
Genetic systems exist for Shewanella spp., and their aerobic growth strongly simplifies
cultivation in comparison to other model strains for cathodic conversions (68). Once its
EET mechanism for electron uptake is well understood, our Shewanella strain could

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become an interesting candidate for genetic engineering toward a strain producing
valuable end products on cathodes.

MATERIALS AND METHODS

Strains and growth conditions. Shewanella strain 4t3-1-2LB and Acetobacterium strain 73-4-6p-4
were previously isolated from acetogenic enrichments, started from freshwater anoxic corrosion prod-
ucts with Fe(0) as the sole electron donor and CO2 and water as the only possible electron acceptors
(Philips et al., submitted). Isolates were obtained by a novel plating procedure using agar plates with an
Fe(0) powder top layer. Shewanella strain 4t3-1-2LB is highly similar to S. fodinae (99% identity, based on
the near-full-length 16S rRNA gene sequence) (NCBI accession number MG835274) (Fig. 1), while
Acetobacterium strain 73-4-6p-4 is closely related to A. wieringae and A. malicum (both 99% identity)
(NCBI accession number MG835272) (Philips et al., submitted). Frozen stocks (⫺80°C, in 10% [vol/vol]
DMSO) of Shewanella strain 4t3-1-2LB were revived in aerobic Luria-Bertani (LB) medium, while anaerobic
DSMZ 135 medium with 1 g · liter⫺1 fructose was used to revive Acetobacterium strain 73-4-6p-4. Cultures
were transferred once and were used in late log phase for inoculation (2.5%, [vol/vol] for all experiments).
Before inoculation, cells were washed by centrifugation and brought into defined medium to avoid
carryover of organic substrates. Washing of Acetobacterium cells was performed inside an anaerobic
chamber (GP-Campus; Jacomex) with an N2-CO2 (90:10, vol/vol) atmosphere.
Corrosion experiments. Experiments were performed in 120-ml serum bottles with 40 ml anaerobic
defined medium and 2 g Fe(0) powder (99%; Riedel-deHaën ⬍212 ␮m, gray color]). The defined medium was
composed as described elsewhere (Philips et al., submitted). The medium was made anoxic by boiling and
N2-CO2 (90:10, vol/vol) gas sparging (at least 30 min) before it was brought under N2-CO2 gas flow into bottles
with Fe(0) powder. This procedure prevented the oxidation of Fe(0) to Fe(III) upon exposure to oxygen and
water. Once in the bottles, the medium was flushed for an additional 10 min. Bottles were autoclaved with
the Fe(0) powder in the medium. Filter-sterilized 3-(N-morpholino)propanesulfonic acid (MOPS) (50 mM,
brought to pH 7.2 with 10 M NaOH) and NaHCO3 (20 mM) were added after autoclaving. Bottles with iron
powder were flushed with sterile N2-CO2 gas for 5 min just before the start of experiments to remove any
hydrogen abiotically formed during the storage of the bottles. Sodium fumarate, succinate, or malate was
added from filter-sterilized anoxic stock solutions at concentrations of 20 mM.
A first experiment tested fumarate conversion by Shewanella strain 4t3-1-2LB in the presence and
absence of Fe(0) (see Fig. S1A in the supplemental material). In addition, abiotic controls with Fe(0) and
fumarate, succinate, or malate were tested. A second series of experiments explored the corrosion-
enhancing mechanism of strain 4t3-1-2LB (Fig. S1B). Medium was exchanged by replacing the liquid
phase with 40 ml fresh anaerobic defined medium, while the iron powder was retained in the bottle.
Afterwards, the bottles were briefly flushed with sterile N2-CO2 gas. The collected medium (spent defined
medium) was filter sterilized (twice through a 0.2-␮m sterile syringe filter) and added to new bottles
containing 5 ml anaerobic defined medium and 2 g Fe(0) powder. In addition, filter-sterilized supernatant
of strain 4t3-1-2LB aerobically grown in LB medium (spent LB medium) was added at 2.5% (vol/vol) to
bottles with 40 ml anaerobic defined medium and 2 g Fe(0) powder, after which the bottles were briefly
flushed. Electrochemical measurements on filtered liquid samples were performed using a rotating-disk
electrode, as described elsewhere (Philips et al., submitted). Iron powder samples were prepared for
scanning electron microscopy (SEM) (69) and visualized with a Phenom Pro SEM (70). A third experiment
investigated Fe(0) corrosion by cocultures of Shewanella strain 4t3-1-2LB and Acetobacterium strain
73-4-6p-4 (Fig. S1C). Both strains were inoculated with 2.5% (vol/vol).
All experiments were performed in triplicate with static incubation at 28°C. Samples were collected
once or twice a week for analysis of the headspace gas composition and VFA and dissolved Fe(II)/Fe(III)
concentrations.
Chemical analyses. The H2 and CO2 contents of gas samples (1 ml) were measured on a compact
gas chromatograph (GC), as previously described (71). Liquid samples (1 ml, filtered through a 0.2-␮m
syringe filter [Chromafil Xtra]) were prepared for VFA or Fe(II)/Fe(III) analysis as reported elsewhere
(Philips et al., submitted). VFA concentrations were measured on an ion chromatograph (72). Additional
standards were prepared from sodium fumarate, malate, and succinate. Dissolved Fe(II)/Fe(III) concen-
trations were measured with an adapted phenanthroline assay (Philips et al., submitted). After the
absorbance of the initial color reaction product was measured [dissolved Fe(II) concentration], a spatula
tip of ascorbic acid powder was added to reduce Fe(III) to Fe(II), and the absorbance was measured again
[dissolved Fe(II) ⫹ Fe(III) concentration]. The pH was measured with an InLab Semi-Micro pH electrode
(Mettler Toledo), and optical densities were measured at 610 nm using a UV-visible spectrophotometer
(Isis 9000; Dr Lange). To allow comparison, all hydrogen, fumarate, malate, succinate, acetate, and
dissolved Fe(II) concentrations were expressed as 10⫺3 moles electron equivalents [assuming two
electrons for each molecule of H2, fumarate, malate, succinate, and Fe(II) and eight electrons for each
molecule of acetate] per volume of the liquid phase (meeq liter⫺1). As hydrogen has a low solubility

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Metallic Iron as an Electron Donor for Shewanella Applied and Environmental Microbiology

(Henry coefficient of 7.8 · 10⫺4 M · atm⫺1 [73]), only its concentration in the headspace was considered
in the calculations. Statistical tests were performing using SPSS Statistics.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AEM
.01154-18.
SUPPLEMENTAL FILE 1, PDF file, 2.0 MB.

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ACKNOWLEDGMENTS
J.P. was funded by a postdoctoral grant from the Special Research Fund (BOF) of
Ghent University. J.P., A.P., J.B.A.A., and K.R. were supported by the European Research
Council via Starter Grant no. 310023 “ELECTROTALK.” K.D.P. was supported by the
Research Foundation Flanders (SBO BRANDING) and the Special Research Fund (BOF)
Concerted Research Actions (GOA, BOF12/GOA/008) from the Flemish Government. In
addition, this project was supported by a King Abdullah University of Science &
Technology (KAUST) Competitive Research Grant (OSR-2016-CRG5-2985-01).
We thank Jana De Bodt (Center for Microbial Ecology and Technology, Ghent
University) for her help with sampling. We acknowledge Greet Van de Velde (Center for
Microbial Ecology and Technology, Ghent University) for performing the ion chroma-
tography analyses. We thank Pieter Candry and Jo De Vrieze (Center for Microbial
Ecology and Technology, Ghent University) for the maintenance of the compact GC.
SEM imaging was performed with the appreciated help of Silvia Hidalgo Martinez and
Filip Meysman (University of Antwerp). We acknowledge Jeet Varia (Center for Microbial
Ecology and Technology, Ghent University) for his useful comments that contributed to
this work.

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