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2018-Novel Shewanella Enhance Corrosion PDF
2018-Novel Shewanella Enhance Corrosion PDF
crossm
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
October 2018 Volume 84 Issue 20 e01154-18 Applied and Environmental Microbiology aem.asm.org 1
Philips et al. Applied and Environmental Microbiology
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).
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.
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
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
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
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,
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
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-
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-
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-
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
(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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