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Applied and Environmental Microbiology, October 2000, p. 4389-4395, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bacterial Phosphating of Mild (Unalloyed)
Steel
Hans-Peter
Volkland,1,2
Hauke
Harms,3
Beat
Müller,4
Gernot
Repphun,5
Oskar
Wanner,1 and
Alexander
J. B.
Zehnder1,2,*
Swiss Federal Institute for Environmental
Science and Technology (EAWAG), CH-8600
Dübendorf,1 Swiss Federal
Institute of Technology (ETHZ), CH-8092 Zurich,2
Swiss Federal Institute of Technology (EPFL), CH-1015
Lausanne,3 Limnological Research Center,
EAWAG, CH-6047 Kastanienbaum,4 and Paul
Scherrer Institute (PSI), CH-5292 Villigen,5
Switzerland
Received 9 March 2000/Accepted 3 August 2000
 |
ABSTRACT |
Mild (unalloyed) steel electrodes were incubated in
phosphate-buffered cultures of aerobic, biofilm-forming
Rhodococcus sp. strain C125 and Pseudomonas
putida mt2. A resulting surface reaction leading to the formation
of a corrosion-inhibiting vivianite layer was accompanied by a
characteristic electrochemical potential (E) curve. First, E increased
slightly due to the interaction of phosphate with the iron oxides
covering the steel surface. Subsequently, E decreased rapidly and after
1 day reached 
510 mV, the potential of free iron, indicating the
removal of the iron oxides. At this point, only scattered patches of
bacteria covered the surface. A surface reaction, in which iron was
released and vivianite precipitated, started. E remained at 510 mV
for about 2 days, during which the vivianite layer grew steadily. Thereafter, E increased markedly to the initial value, and the release
of iron stopped. Changes in E and formation of vivianite were results
of bacterial activity, with oxygen consumption by the biofilm being the
driving force. These findings indicate that biofilms may protect steel
surfaces and might be used as an alternative method to combat corrosion.
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INTRODUCTION |
Due to the poor corrosion resistance
of mild (unalloyed) steel, virtually all items made of this material
have to be protected against corrosion. The most common protection
method is phosphating, i.e., coating the steel surface with the
phosphates of zinc, iron, or manganese (34). This procedure
is carried out at temperatures up to 95°C and pH values between 2 and
3.5 (21). Media used for phosphating normally contain high
concentrations of zinc (in the range of several grams per liter) or
manganese and also contain accelerators like nitrate, nitrite,
chlorate, peroxides, and organic nitrocompounds (31). During
phosphating, a considerable amount of heavy metal sludge is formed and
must be removed. Several attempts have been made to develop alternative
methods that are less toxic to the environment.
Pedersen et al. (17) showed that Pseudomonas sp.
strain S9 and Serratia marcescens sp. strain EF 190 can
decrease the corrosion rate of mild steel when applied as dense
suspensions (109 ml
1) or as living biofilms
(17-19). A protective effect of Pseudomonas fragi and Escherichia coli DH5 was found by Jayaraman
et al. (13). Here, the formation of a biofilm was crucial,
as oxygen depletion under the biofilm was responsible for the corrosion
protection (12). However, the mechanical instability of
biofilms was seen as a drawback for their technical application.
In a recent study (32), we showed that growing the aerobic
biofilm-forming bacteria Rhodococcus sp. strain C125 and
Pseudomonas putida mt2 in mineral medium containing more
than 2 mM phosphate induced a surface reaction on mild steel coupons,
resulting in the formation of vivianite. Vivianite, a barely insoluble
iron(II) phosphate, is one of the compounds formed in technical acidic phosphating and is known for its corrosion protective effect. The
biologically vivianite-coated steel coupons showed good corrosion protection even after removal of the biofilms. Here we report on the
electrochemical mechanism of vivianite formation by biofilms of
Rhodococcus sp. strain C125 and P. putida mt2.
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MATERIALS AND METHODS |
Bacteria and media.
Rhodococcus sp. strain C125
(25) and P. putida mt2 (33) were grown
aerobically at room temperature either in nutrient broth (NB) (8 g of
dry NB liter of distilled H2O1 Biolife,
Milan, Italy) or in a mineral medium (32) containing either
6 mM sodium benzoate or ethanol as the sole source of carbon and energy
and in 20 mM phosphate buffer (2.7 g of KH2PO4
liter1, adjusted to pH 7.2 with NaOH. The same 20 mM
phosphate buffer was used for flushing steel coupons and as medium for
resting cell suspensions of Rhodococcus sp. strain C125. The
preparation of supernatants from 2-day-old cultures of
Rhodococcus sp. strain C125 in mineral medium containing 6 mM benzoate has been described before (32).
Steel coupons.
Square coupons of mild steel (steel 37/AISI
10-18) (20 by 20 by 2.5 mm) were used. For experiments involving atomic
force microscopy (AFM), circular coupons with a diameter of 14 mm were used. A 1.5-mm-diameter hole served to fix coupons to a
nonbiodegradable polymeric thread. The coupons were polished with P1000
silicon carbide paper (SIA, Frauenfeld, Switzerland) to obtain a
uniform smooth surface, degreased in acetone, and washed with ethanol.
Electrochemical measurements.
For measurements of E, steel
electrodes were prepared such that the noninsulated end of a
plastic-coated wire was connected to the hole of the steel coupon. The
connection was embedded in a silver-containing epoxy resin (Epo-Tek
A/B; Polyscience AG, Cham, Switzerland), and after solidification of
the resin at 75°C for 2 h, the connection was insulated with an
electrical coating (Scotchkote, 3M, St. Paul, Minn.). The steel
electrode was connected to a silver-silver chloride reference electrode
(Orion, Beverly, Mass.) via a voltmeter with high resistance.
Measurements were made automatically every 30 to 120 s. The E
values were converted to standard hydrogen electrode potentials by
adding 211 mV, expressed as mVSCE (standard calomel electrode).
Electrodes were placed in 300-ml Erlenmeyer flasks filled with 250 ml
of the desired medium. The flasks were inoculated with 0.5% of a
24-h-old preculture cultivated in this medium and incubated for 5 days
at 25°C. Aerobic conditions were maintained using a magnetic stirrer.
For experiments in which the temperature was varied, a completely
filled 1-liter bioreactor (Schmid, Zofingen, Switzerland) was used.
To determine the effect of the absence of direct contact between
bacteria and the steel surface, the electrodes were enclosed
in a
membrane with an exclusion size of 10,000 to 12,000 (molecular
weight)
(Medicell, London, Great Britain) to prevent access of
the growing
Rhodococcus sp. strain C125 to the steel surface.
When
biofilms had to be removed, the electrodes were flushed thoroughly
with
20 ml of 20 mM phosphate buffer using a syringe. Control
experiments
were performed with electrodes immersed in Erlenmeyer
flasks (250 ml)
under various conditions, namely, stirred sterile
medium containing
benzoate or sterile culture supernatant for
5 days or in sterile,
anaerobic or microaerophilic benzoate-containing
medium for 2 days.
Anaerobic or microaerophilic conditions were
maintained by gassing the
medium for 2 days with N
2 or a mixture
of 98%
N
2 and 2% O
2, respectively. Afterward, gassing
was stopped
and the medium was stirred for 2 more days in
air.
To measure E, which yields information on reactions at a surface, and
the polarization resistance
Rp of surface
layers, which
yields information on the structure of surface layers, we
used
electrochemical impedance spectroscopy (EIS). The results from
these measurements can be used to calculate the corrosion current
Icorr as a measure of the corrosion rate. The
setup for EIS consisted
of a potentiostat (PC3/300 potentiostat with
CMS 100/105 system;
Gamry Instruments Inc., Warminster, Pa.). The
reference electrode
was the same silver- silver chloride electrode used
for the potential
measurements, the counter electrode was a Pt wire,
and the steel
electrode acted as a working electrode. Medium containing
benzoate
was incubated with
Rhodococcus sp. strain C125 in a
300-ml Erlenmeyer
flask. Stirring was performed using a magnetic
stirrer bar powered
by an air pressure rotor to avoid electrical noise.
By using a
program routine of the personal-computer-controlled system,
E
and
Rp were measured automatically. The
Rp was calculated from
the slope of a
slow-current voltage curve (E
7 mV < E < E +
7 mV,
dE/dt = 2 mV/s). The
Icorr
was estimated from the equation
Icorr =
babc/[(
ba +
bc)2.3
Rp], with
ba and
bc being the Tafel
coefficients for the anodic and cathodic current, respectively,
which
were determined by usual Tafel analysis of current-voltage
characteristics in the phosphate buffer at the blank steel surface
to
be 0.4 ± 0.05 V/decade and 0.87 ± 0.05 V/decade,
respectively
(
3). As the resistance has to be kept high and
constant for
the measurement of E,
Rp cannot be
measured simultaneously with
the experimental setup
described.
Analytical procedures.
Dissolved Fe(II) was determined
photometrically after complexation with FerroZine (10) using
a UV-visible light photometer (model U-1100; Hitachi Tokyo, Japan) at
562 nm. Dissolved Fe(II) and Fe(III) concentrations were determined
after filtration of the solutions (pore size, 0.2 µm; Schleicher & Schuell, Dassel, Germany); total iron concentration was determined
without filtration. Fe(III) and total iron were determined as Fe(II)
after 3 min of reduction with a 4% solution containing 208.5 g of
hydroxylamine hydrochloride liter1 in 12% hydrochloric
acid. The optical density at 546 nm (OD546) of bacterial
suspensions was measured photometrically. Oxygen concentrations in the
growth medium were monitored with an oxygen electrode (Mettler Toledo,
Urdorf, Switzerland) which remained in the growth medium during the
entire experiment. Dissolved organic carbon (DOC) was measured with a
carbon analyzer (model 5000A; Shimadzu, Tokyo, Japan).
Surface analysis.
Scanning electron microscopy (SEM) was
used to analyze the steel coupon surface. The coupons were washed with
double-distilled water, air dried, coated with carbon in a Balzers
carbon thread evaporating device (CED) 010 (Balzers, Balzers,
Liechtenstein), and examined at an acceleration voltage of 20 kV with a
Philips XL-30 SEM (Philips, Eindhoven, The Netherlands) equipped with a
LaB6 electron source. The SEM was combined with an
energy-dispersive X-ray system (SEM-EDAX), in which acceleration
voltages of 10 to 20 kV were used. To measure the thickness of the
vivianite layer, coupons were embedded in epoxy resin (Epofix; Struers, Copenhagen, Denmark). Then, a cross-section was made in the center of
the coupon and polished with diamond dust of 1-µm grain size. After
carbon coating, the samples were measured in the SEM-EDAX with
acceleration voltages of 2 to 5 kV using a back-scattering electrode.
AFM was used to obtain images with higher resolution. AFM images were
made after washing the coupon with double-distilled water in a
Nanoscope II (Digital Instruments, Santa Barbara, Calif.) operated with
a 12-µm scanner in contact mode, which means that soft materials like
bacteria or organic polymers are wiped away. Silicon high-aspect ratio
tips with a cone angle of approximately 10° and a tip radius of less
than 10 nm (Nanosensors, Wetzlar, Germany) were used. Biofilms on steel
coupons were investigated by epifluorescence microscopy after staining
with acridine orange (32). X-ray diffraction measurements
were performed using Cu-K
radiation (32).
X-ray photoelectron spectroscopy (XPS) was used to examine crystals for
their in-depth homogeneity. This method which can
provide information
to a depth of about 3 nm was applied because
of the small size of the
crystal surfaces. The accuracy of the
concentration determination is
typically in the range of ±3 to
5 atom%. Measurements were performed
on a PHI Quantum 2000 instrument
with a lateral resolution of about 10 µm. This permitted the unambiguous
analysis of the crystal
surface alone. The sputter depth profiling
was performed with argon
ions at 3 keV. The sputter rate was calibrated
for SiO
2
being 18 nm/min. The concentration profiles of iron (Fe
2p), oxygen (O
1s), carbon (C 1s), phosphorous (P 2s) and calcium
(Ca 2p) in depths of
0 to 35 nm were recorded using the most-intensive
core level lines of
the respective
elements.
| |
RESULTS |
E.
When the coupons were incubated in cultures of
Rhodococcus sp. strain C125 growing in 6 mM benzoate, E of
the coupon surface immediately increased and reached the first maximum
(Emax1) of 160 mV after about 10 h (Fig. 1, curve
A). Thereafter, E decreased slowly for
another 20 to 40 h before dropping within 2 min from about +80 mV
to a minimum (Emin) of 510 mV, where it remained for
the next 40 to 50 h. Subsequently, E suddenly increased and then
gradually leveled off at a new maximum, Emax2, after
100 to 140 h. Incubations in 10 or 15 mM benzoate resulted in
similar curves (data not shown) with E also reaching 510 mV but
remaining longer at this low level. During incubation in cultures of
Rhodococcus sp. strain C125 growing in 2 mM benzoate, the
potential curve (Fig. 1, curve B) first followed curve A, dropped to 0 mV after 18 h, returned rapidly to a level of +110 mV, and finally
slowly increased. Potential curves of incubations with 1 or 0.5 mM
benzoate were similar to those with 2 mM but had less-pronounced
minima. In sterile medium containing 6 mM benzoate (Fig. 1, curve C) or phosphate buffer, E immediately increased, followed by a slower further
rise.
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FIG. 1.
Time course of E during incubation with
Rhodococcus sp. strain C125 in 6 mM benzoate medium (curve
A), 2 mM benzoate medium (curve B), or sterile 6 mM benzoate medium
(curve C). mVSCE, standard hydrogen electrode potential.
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The significance of the biofilm at the steel surface for the
development of E was studied by removing the biofilm by flushing
or
slowing its activity down by cooling. Removal of the biofilm
immediately after E dropped made E increase slowly to about
400
mV.
Once the coupons were put back in the medium, they corroded,
forming
brown iron oxides. Removal of the biofilm 1 day after
the drop in E
resulted in an increase of E to E
max2 similar to
that in
curve A of Fig.
1. When the growth medium was cooled from
25 to 2°C,
E increased immediately (Fig.
2).
Subsequent heating
to 25°C made E decrease to E
min again.
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FIG. 2.
Effect of temperature change on E during incubation with
Rhodococcus sp. strain C125 in 6 mM benzoate medium.
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Incubation in a culture of
P. putida mt2 growing on 6 mM
benzoate resulted in potentials similar to those in curve A of Fig.
1
except that the drop in E occurred after 15 to 20 h. When ethanol
was used as the carbon source, results similar to those found
with
benzoate were obtained for both
Rhodococcus sp. strain C125
and
P. putida mt2. E
min was
510 mV and +40 mV
with 6 and 2 mM
ethanol, respectively. Immersion of steel electrodes
(i) into
resting cell suspensions of either strain in phosphate buffer,
(ii) into sterile-filtered culture supernatants, or (iii) into
an
actively growing culture with the electrode enclosed in a dialysis
membrane all led to potential curves similar to curve B in Fig.
1.
Rp and Icorr.
Electrochemical impedance spectroscopy (EIS) measurements performed to
determine the development of E, Rp, and
Icorr during incubation with
Rhodococcus sp. strain C125 in medium containing 6 mM
benzoate showed that Rp strongly fluctuated for
the first 50 h and decreased to almost 0 
cm2 by
the time E dropped (Fig. 3A). Then, it
remained stable and increased when E returned to Emax2.
Icorr was negligible initially, strongly
increased when E dropped to Emin, and then gradually decreased again (Fig. 3B and C). During the rise of E to
Emax2, Icorr transiently increased
again but dropped to the initial value.
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FIG. 3.
Time course of EIS measurements showing E and
Rp (A), E and corrosion current
Icorr (B), and details of all three
determinations during Emin (C). Incubation were made with
Rhodococcus sp. strain C125 in 6 mM benzoate medium.
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During incubation with
Rhodococcus sp. strain C125 in medium
containing 1 mM benzoate instead of 6 mM benzoate,
Rp remained
permanently unstable and
Icorr stayed low (data not shown). The
same
results were obtained in the absence of bacteria. Since the
measurement
of
Rp influences the corrosion potential by only
a
few millivolts (
3), the observed outcome could be
expected.
Surface analysis.
The appearance of the coupon surface before
incubation was investigated by SEM (Fig.
4A). X-ray diffraction analysis of
coupons which had been incubated with Rhodococcus sp. strain
C125 growing on 6 mM benzoate for 120 h showed vivianite
[Fe3(PO4)2 · 8H2O] as the only crystalline compound on the coupons'
surface (32). No vivianite was detected after incubation of
coupons in sterile, benzoate-containing mineral medium, sterile
phosphate buffer, sterile culture supernatant, resting cell
suspensions, or cultures of Rhodococcus sp. strain
C125 growing on 2 mM or less benzoate.
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FIG. 4.
Surface of mild steel coupon as observed by SEM before
incubation with P. putida mt2 in 6 mM benzoate medium (A)
and AFM directly after breakdown of the potential to Emin
(B). In panel B, areas with crystalline (cr) and amorphous (am)
material can be clearly distinguished from unaltered surface (un).
Incubations with Rhodococcus sp. strain C125 gave very
similar results.
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AFM analysis of an incubated coupon 1 min after E had dropped revealed
a partial surface coverage with amorphous and crystalline-looking
material (Fig.
4B). At this point, the surface was only sparsely
covered with mostly single bacteria and small aggregates. SEM-EDAX
of a
cross-section of a coupon after 48 h of incubation could
visualize
the unaltered steel which was covered by a layer of
vivianite 2 to 3 µm thick (Fig.
5). Incubation in
cultures growing
on 2 mM or less benzoate or in sterile benzoate media
left coupon
surfaces unaltered.
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FIG. 5.
Cross-section analyzed with SEM-EDAX of a coupon
incubated for 2 days. The vivianite layer is about 2 µm thick. The
insert shows the atomic percentage of iron and phosphorus found in the
steel, the vivianite layer, and the epoxy resin (indicated by 1, 2, and
3, respectively). This sample was obtained with P. putida
mt2 in 6 mM benzoate medium. Incubations with Rhodococcus
sp. strain C125 gave comparable results.
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Characteristics of the culture medium during coupon
incubation.
The time courses of suspended biomass formation
(OD546), DOC as a measure of substrate consumption, and
oxygen saturation in a culture of Rhodococcus sp. strain
C125 growing on 6 mM benzoate are shown in Fig.
6. When E dropped, the OD546
had reached 0.4 and O2 was reduced to 10% of the initial
air saturation value. E began to rise again by the time the culture
became stationary because benzoate had been used up and the dissolved
O2 began to increase back to air saturation values.
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FIG. 6.
Time course for optical density (OD546),
oxygen concentration ([O2] in percent saturation
concentration), and DOC (in percent initial concentration) during
incubation with Rhodococcus sp. strain C125 in 6 mM benzoate
medium. E is also shown for comparison.
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An E curve similar to that in curve A of Fig.
1 was observed when
electrodes were immersed into benzoate-containing medium
which had been
completely deaerated by flushing with nitrogen,
whereas gassing with
2% oxygen and 98% nitrogen resulted in an
E curve similar to that in
curve B of Fig.
1. This clearly shows
that an oxygen partial pressure
of 0.02 atm was not low enough
to induce E to
drop.
The concentrations of Fe(II) [Fe
2+], Fe(III)
[Fe
3+], and the total iron concentration were low
initially (Fig.
7). After E decreased,
the concentrations of all iron species increased steadily and
reached
maximum values when E increased again. Afterward [Fe
2+]
decreased, while [Fe
3+] remained constant. Throughout
their release into the medium,
Fe
3+ and Fe
2+
were present at a constant ratio. The maximum iron concentration
in the
medium in this particular experiment was 0.17 mM but varied
between 0.1 and 1.0 mM in independent experiments. Most of the
iron was present in
soluble form, since filtration through a 0.2-µm-pore-size
filter removed less than 5%.
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FIG. 7.
Concentration of Fe2+, Fe3+, and
total Fe during incubation with Rhodococcus sp. strain C125
in 6 mM benzoate medium. The Fe2+/Fe3+ ratio
during Emin remained constant at 2:1.
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Reproducibility and generalization.
The data in the figures
are from representative experiments. The variability (n 
5) for E in the cultures of Rhodococcus sp. strain C125
and P. putida mt2 in the medium with 6 mM benzoate measured
was +160 mV ± 20 mV for Emax1, 510 mV ± 5 mV
for Emin, and +170 mV ± 20 mV for Emax2
(Fig. 8). The time course of E depended on the medium (substrate concentration), the organism, and its growth
characteristics (lag phase and growth rate). For Rhodococcus sp. strain C125 (n = 8), the drop in E typically
occurred between 30 and 50 h (compare Fig. 1 and 3),
Emin lasted for 50 to 60 h, and Emax2 was
reached after 100 to 140 h. The higher growth rate of P. putida mt2 (n = 5) resulted in a drop in E after
15 to 20 h, an Emin of 25 to 30 h, and an
Emax2 after 60 to 80 h. Emin lasted longer
in the presence of higher substrate concentrations for all cases (data
not shown).
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FIG. 8.
Division of the E curve, curve A in Fig. 1, into four
phases and indication of potential minimum and the two maxima.
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E measurements have also been done for cultures of biofilm-forming
Pseudomonas aeruginosa PO1 (ATCC 15692),
P. putida WCS358
(
15),
Pseudomonas fluorescens
p62 (
31), and
E. coli ML 30
(DSM 1329).
E
max1, E
min, and E
max2 for all
pseudomonads and
E. coli were quantitatively the same as for
Rhodococcus sp. strain
C125 and
P. putida mt2,
but the time dependency varied. In all
cases, vivianite precipitation
could be seen and a considerable
corrosion protection be measured
(
32; data not shown). The non-biofilm-forming
Streptomyces pilosus (DSM 40714) did not trigger vivianite
precipitation,
and surface alterations or corrosion inhibition were not
measured
(
32). No characteristic E curve as described here
could be measured
with
S. pilosus.
| |
DISCUSSION |
To facilitate the discussion of the E curve (Fig. 1, curve A)
accompanying the biological phosphating of mild steel, we will divide
the time course into four phases: phase I, increase to Emax1; phase II, decrease to Emin; phase III,
stability at Emin; and phase IV, increase to
Emax2 (Fig. 8).
Phase I.
In phase I, E increases in all incubations in
phosphate-containing media, regardless of the presence of active
bacterial cultures. This increase can be explained by the reaction of
phosphate with the iron oxide layer covering the steel electrode. This
layer typically consists of Fe3O4 and
-Fe2O3 (24). It is only 0.5 to 1 nm thick and of heterogeneous composition and thus provides little
corrosion protection. Phosphate acts as a temporary corrosion inhibitor
at pH > 4 and stabilizes the layer due to its ability to form
binuclear complexes with iron oxides (23, 27-29). This stabilization can be seen as an ennoblement of the electrode, i.e., an
increase in its potential from +60 to +160 mV. Pryor and Cohen
(20) found a similar increase in the potential from +50 to
+150 mV when a steel electrode was immersed into 0.1 N sodium phosphate
of pH 7.0 ± 0.2.
Phase II.
In phase II, E decreased only when a sufficiently
active bacterial culture was present. Direct contact with the steel
coupon used as the electrode was necessary. The very fast drop of E and partial surface coverage with vivianite only 1 min later indicated the
sudden loss of the entire oxide layer and its rapid replacement by an
iron phosphate layer. When bacteria were absent or present in low
density because of limited substrate supply, the drop in E was much
less pronounced or did not occur. The time course of Rp was in agreement with these observations as
it fluctuated as long as the heterogeneous iron oxide layer was present
and became stable as soon as vivianite was detected (Fig. 3A). As
visible in Fig. 4B, surface structures, at least partly, already
consisted of vivianite. Vivianite has been shown to precipitate easily, even out of highly impure solutions like urban sewage sludge
(7). In technical phosphating, the first layer on the iron
surface normally consists of vivianite, since its formation is favored by its epitactic relationship with iron (16).
The question arises, in what way the surface reaction could have been
induced by bacterial activity. Bacterial factors which
are known to
influence steel corrosion so far are the excretion
of metabolites
(
8,
19), specific interactions between the
cell wall and
steel surface (
4,
5), and oxygen consumption
(
9,
12,
14). In our experiments, excretion of bacterial
products can be
excluded as a factor, since filtered culture supernatant
did not alter
the steel surface. Direct specific interactions
between cell walls and
steel surface are unlikely. For bacteria
with very different surface
polymers (hydrophobic mycolic acids
for
Rhodococcus sp.
strain C125 [
6] and hydrophilic polysaccharides
probably covered with proteins for
P. putida mt2
[
22]), the
same effects were observed at the steel
surface.
A more likely explanation is bacterial oxygen consumption directly at
the surface. Pryor and Cohen (
20) found a drop in
E to
520
mV and vivianite formation when they immersed steel
electrodes in
deaerated 0.1 N sodium phosphate solutions of pH
7.0 ± 0.2. In
our own experiments, the bulk liquid of the bacterial
culture, although
stirred, was about 10% O
2 air saturated at the
time of the
drop in E. We assume that O
2 consumption by the few
adhered
bacteria in combination with diffusion limitation may
have reduced the
O
2 concentration locally at the steel surface
sufficiently
for the iron oxide layer to dissolve. First, the
layer may have been
disrupted under single bacteria or biofilm
patches as a result of
oxygen depletion, possibly enhanced by
excretion products. Once small
holes in the oxide layer were formed,
corrosion of the underlying iron,
according to the equation Fe
+ 1/2O
2 + H
2O
Fe(OH)
2, could have added to the
consumption of oxygen
(
28). The steel surface would now have
consisted of two electrochemically
different types of areas: a large
area without biofilm coverage
where E was still high and a smaller area
with few biofilm-covered
spots where E was already at E
min.
However, one would expect to
find no or little chemical reactivity at
such a surface. Moreover,
this view is in contrast with Fig.
4B, which
showed that the surface
was already highly covered with inorganic
material other than
the iron
oxides.
Since the growth medium did not remove the oxide layer, we tested the
possibility that biofilm-covered areas electrochemically
influenced
uncovered areas. Therefore, an electrode at an E, which
had just
dropped due to incubation with
P. putida mt2 in 6 mM
benzoate medium, was connected to a second, still oxidized electrode
that was enclosed in a membrane filled with sterile medium. The
electrode with the low potential was supposed to simulate areas
covered
with a biofilm, and the second electrode uncovered areas.
Both
electrodes were placed into the bacterial suspension. E of
the
membrane-enclosed electrode dropped immediately to E
min,
and
both electrodes remained at E
min for an extended time.
This strongly
indicates a self-supporting corrosion process that
rapidly removed
the entire oxide layer and replaced it by vivianite.
Vivianite
has a very low solubility product
Ksp
(
2):
The chemical reaction and dissociation constants for the phosphate
ions involved (
1) are as follows:
At pH = 7.2 [PO
43] is
10
7.15 M, i.e., the medium is already oversaturated at
less than 10
7 M iron(II). The corrosion process shifts
the pH at the surface
to higher values (
28) and consequently
to higher PO
43 concentrations. Therefore,
saturation is reached at even lower
iron(II) concentrations. The iron
concentration at the time of
the drop in E can roughly be estimated
from
Icorr at this time.
The amount
N
of iron released is
N =
Icorr /
(
n ×
F), where
n is the valence
of the iron species (here 2 for Fe
2+) and
F is
Faraday's constant (96,485 A s mol
1). With
Icorr of 1.94 mA cm
2, as much as
6.0 × 10
7 mol cm
2 of iron must have
been released within 1 min. As the reactive
electrode surface was 7 cm
2 and the volume was 250 ml, the iron concentration could
have
been 17 µM after 1 min, meaning that the medium was already
highly
oversaturated with respect to vivianite at that time. Therefore,
vivianite precipitation within seconds after the potential drop
is very
likely.
Phase III.
During phase III, E remained relatively stable at
Emin for 50 to 60 h. Only a slight increase from 510
to 490 mV could be seen. Removal of the biofilm in the very beginning
of this phase hindered the vivianite formation, whereas biofilm removal
after 24 h had no such effect. Therefore, we assume that in the
first hours after E dropped, the crystallization process proceeded and could be easily disturbed, with the consequence that the vivianite layer did not fully develop. The fast growth of the vivianite layer is
also reflected by the strong increase in Rp of
the steel surface (Fig. 3B). Between 15 and 30 h after the drop in
E, the initial crystallization processes seemed to have ended and a 2- to 3-µm-thick vivianite layer covered the entire surface (Fig. 5).
Increase of Rp and decrease of
Icorr (Fig. 3C) indicate a slowdown of both
vivianite layer growth and corrosion rate. To keep E low, bacterial
activity was necessary. Removal of the biofilm after 48 h of
incubation resulted in an increase of E to Emax2, as did
reduction of the temperature from 25 to 2°C. Subsequent heating to
25°C lowered E again to Emin (Fig. 2). The fact that the
potential reacted so fast to temperature changes indicate that it was
controlled by the oxygen concentration near the steel surface as a
result of biofilm activity and not by an interaction of excreted
metabolites or bacterial surface polymers with the steel surface.
Phase IV.
The rise in E in this phase was paralleled by the
increase of the oxygen partial pressure (Fig. 6), a fast rise in
Rp (Fig. 3A), a drop in
Icorr (Fig. 3B), and a drop in the
Fe2+ concentration (Fig. 7). The addition of 6 mM benzoate
lowered E again to Emin, probably due to the stimulation of
oxygen consumption by the bacteria. This suggests that there were still
pores in the vivianite layer at the end of phase III (26).
While bacterial oxygen consumption had ceased, iron at the bottom of
the pores had probably been oxidized and given rise to an increase in
E. After consumption of the newly added benzoate, E rose again. Further benzoate additions did not lower E anymore (data not shown), indicating that the ongoing vivianite formation now might have sealed the pores to
sizes rendering the access of redox reactive molecules to the bottom of
the vivianite layer impossible. The value of +170 mV for
Emax2 was in the same range as Emax1. Thus, it
can be assumed that vivianite, being very sensitive to oxidation of its
surface (11), became oxidized. Such a surface reaction
explains a temporary rise in Icorr and
Rp. The assumption is further supported by the
blue color of oxidized vivianite (11) found after extensive incubation (32). The oxide layer must have been thin, since no significant change in the proportion of the elements Fe, P, and O
present in vivianite could be measured by XPS of the outer vivianite
layer (data not shown). Only a thin layer of organic compounds, which
probably consisted of bacterial polymers, was found to cover the
vivianite. Since the exact stoichiometry of oxidized vivianite,
presumably a mixed iron (Fe2+ and Fe3+)
phosphate oxide, is not known, it remains unclear whether such a
compound could have been detected by XPS at all.
Our investigations have shown that biofilms, which are commonly assumed
to cause biocorrosion, can be used for corrosion prevention
by
phosphating a mild steel
surface.
| |
ACKNOWLEDGMENTS |
This work was supported financially by the Swiss Federal Office
of Education and Science.
We thank Stefan Hug and Peter Weidler for assistance with AFM and SEM,
Peter Lienemann for X-ray analysis, and Roland Hauert and Joerg
Patscheider for SEM-XPS.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Swiss Federal
Institute for Environmental Science and Technology (EAWAG),
Überlandstrasse 133, CH-8600 Dübendorf, Switzerland. Phone:
41 1 823 5001. Fax: 41 1 823 5398. E-mail: zehnder{at}eawag.ch.
| |
REFERENCES |
| 1.
|
Abbott, G. A., and W. C. Bray.
1909.
Ionization relations of ortho- and pyrophosphoric acids and their sodium salts.
J. Am. Chem. Soc.
31:729-736[CrossRef].
|
| 2.
|
Al-Borno, A., and M. B. Tomson.
1994.
The temperature dependence of the solubility product constant of vivianite.
Geochim. Cosmochim. Acta
58:5373-5378[CrossRef].
|
| 3.
|
American Society for Metals.
1992.
ASM handbook, vol. 13.
ASM International, Materials Park, Ohio.
|
| 4.
|
Beech, I. B., and C. C. Gaylarde.
1991.
Microbial polysaccharides and corrosion.
Int. Biodeterior.
27:95-107.
|
| 5.
|
Beech, I. B.,
V. Zinkevich,
R. Tapper, and R. Gubner.
1998.
Direct involvement of an extracellular complex produced by a marine sulphate-reducing bacterium in the deterioration of steel.
Geomicrobiol. J.
15:121-134.
|
| 6.
|
Bendinger, B.,
H. H. M. Rijnaarts,
K. Altendorf, and A. J. B. Zehnder.
1993.
Physicochemical cell surface and adhesive properties of coryneform bacteria related to the presence and chain length of mycolic acids.
Appl. Environ. Microbiol.
59:3973-3977[Abstract/Free Full Text].
|
| 7.
|
Frossard, E.,
J. P. Bauer, and F. Lothe.
1997.
Evidence of vivianite in FeSO4 flocculated sludge.
Water Res.
31:2449-2454[CrossRef].
|
| 8.
|
Gaylarde, C. C., and J. M. Johnsten.
1980.
The importance of microbial adhesion in anaerobic metal corrosion, p. 511-513.
In
R. C. W. Berkely, J. M. Lynch, J. Melling, P. R. Rutter, and B. Vincent (ed.), Microbial adhesion to surfaces. Ellis Horwood Ltd., Chichester, Great Britain.
|
| 9.
|
Geesey, G. G.
1991.
What is biocorrosion?, p. 155-164.
In
H. C. Flemming, and G. G. Geesey (ed.), Biofouling and biocorrosion in industrial water systems. Springer-Verlag, Berlin, Germany.
|
| 10.
|
Gibbs, C. R.
1976.
Characterization and application of FerroZine iron reagent as a ferrous iron indicator.
Anal. Chem.
48:1197-1201[CrossRef].
|
| 11.
|
Gmelin Institute for Inorganic Chemistry.
1988.
Handbook of inorganic and organometallic chemistry, vol. 59. , p. 4B.
Springer-Verlag, Berlin, Germany.
|
| 12.
|
Jayaraman, A.,
E. T. Cheng,
J. C. Earthman, and T. K. Wood.
1997.
Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion.
Appl. Microbiol. Biotechnol.
48:11-17[CrossRef][Medline].
|
| 13.
|
Jayaraman, A.,
J. C. Earthman, and T. K. Wood.
1997.
Corrosion inhibition by aerobic biofilms on SAE 1018 steel.
Appl. Microbiol. Biotechnol.
47:62-68[CrossRef].
|
| 14.
|
Lee, W., and D. de Beer.
1995.
Oxygen and pH microprofiles above corroding mild steel covered with a biofilm.
Biofouling
8:273-280.
|
| 15.
|
Marugg, J. D.,
M. van Spanje,
W. P. M. Hoekstra,
B. Schippers, and P. J. Weisbeek.
1985.
Isolation and analysis of genes involved in siderophore biosynthesis in plant growth-stimulating Pseudomonas putida WCS358.
J. Bacteriol.
164:563-570[Abstract/Free Full Text].
|
| 16.
|
Neuhaus, A., and M. Gebhardt.
1968.
Epitaxy and corrosion resistance of inorganic protective layers on metals, p. 91-119.
In
Interface conversion for polymer coating. Elsevier, New York, N.Y.
|
| 17.
|
Pedersen, A.,
S. Kjelleberg, and M. Hermansson.
1988.
A screening method for bacterial corrosion of metals.
J. Microbiol. Methods
8:191-198[CrossRef].
|
| 18.
|
Pedersen, A., and M. Hermansson.
1989.
The effects on metal corrosion by Serratia marcescens and a Pseudomonas sp.
Biofouling
1:313-322.
|
| 19.
|
Pedersen, A., and M. Hermansson.
1991.
Inhibition of metal corrosion by bacteria.
Biofouling
3:1-11.
|
| 20.
|
Pryor, M. J., and M. Cohen.
1951.
The mechanism of the inhibition of the corrosion of iron by solutions of sodium orthophosphate.
J. Electrochem. Soc.
98:263-271.
|
| 21.
|
Rausch, W.
1988.
Die Phosphatierung von Metallen.
Leuze Verlag, Saulgau, Germany.
|
| 22.
|
Rijnaarts, H. H. M.,
W. Norde,
J. Lyklema, and A. J. B. Zehnder.
1995.
The isoelectric point of bacteria as an indicator for the presence of cell surface polymers that inhibit adhesion.
Colloids Surf. B
4:191-197[CrossRef].
|
| 23.
|
Rose, J. M.,
A. M. Flank,
A. Mason,
J. Y. Bottero, and P. Elmerich.
1997.
Nucleation and growth mechanisms of Fe oxohydrides in the presence of PO4 ions.
Langmuir
13:1827-1834[CrossRef].
|
| 24.
|
Sato, N.,
K. Kudo, and T. Noda.
1975.
Anodic passivating films on iron in phosphate and borate solutions.
Z. Phys. Chem.
98:271-284.
|
| 25.
|
Schraa, G.,
B. M. Bethe,
A. R. W. van Neerven,
W. J. J. van den Wende, and A. J. B. Zehnder.
1987.
Degradation of 1,2-dimethylbenzene by Corynebacterium strain C125.
Antoine Leeuwenhoek
53:159-170.
|
| 26.
|
Schultze, J. W., and A. Losch.
1991.
A new electrochemical method for the determination of the free surface of phosphate layers.
Appl. Surf. Sci.
52:29-38[CrossRef].
|
| 27.
|
Sinniger, J.
1992.
Die Inhibition der Auflösung von Eisen (III) (hydr)oxiden und ihre Beziehung zur Passivität von Eisen. Ph.D. thesis, p. 9893.
ETH, Zürich, Switzerland.
|
| 28.
|
Stumm, W.
1998.
Corrosion of metals in aquatic systems; an introduction. EAWAG schriftenreihe 12.
EAWAG, Dübendorf, Switzerland.
|
| 29.
|
Szklarska-Smialowska, S., and R. W. Staehle.
1974.
Ellipsometric study of the formation of films on iron in orthophosphate solutions.
J. Electrochem. Soc.
121:1393-1401.
|
| 30.
|
van Loosdrecht, M. C. M.,
J. Lyklema,
W. Norde, and A. J. B. Zehnder.
1989.
Bacterial adhesion: a physicochemical approach.
Microb. Ecol.
17:1-16.
|
| 31.
|
VCH.
1990.
Ullmann's encyclopedia of industrial chemistry, vol. A16. Metals, surface treatment.
VCH, Weinheim, Germany.
|
| 32.
|
Volkland, H. P.,
H. Harms,
K. Knopf,
O. Wanner, and A. J. B. Zehnder.
2000.
Corrosion inhibition of mild steel by bacteria.
Biofouling
15:287-297.
|
| 33.
|
Williams, P. A., and J. W. Worsey.
1976.
Ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new TOL plasmids.
J. Bacteriol.
125:818-828[Abstract/Free Full Text].
|
| 34.
|
Wranglén, G.
1985.
Korrosion und Korrosionsschutz.
Springer-Verlag, Berlin, Germany.
|
Applied and Environmental Microbiology, October 2000, p. 4389-4395, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
![K<SUB><UP>sp</UP></SUB><UP> = </UP>[<UP>Fe<SUP>2+</SUP></UP>]<SUP><UP>3</UP></SUP> [<UP>PO<SUB>4</SUB><SUP>3−</SUP></UP>]<SUP><UP>2</UP></SUP><UP> = 10<SUP>−36</SUP> mol<SUP>5</SUP> liter<SUP>−5</SUP></UP>](/content/vol66/issue10/fulltext/4389/img001.gif)
