878 DOI: 10.1002/maco.200905582 Materials and Corrosion 2011, 62, No.

Corrosion of API XL 52 steel in presence of Clostridium

celerecrescens
O. A. Ramos Monroy, M. J. Hernández Gayosso*, N. Ruiz Ordaz,
G. Zavala Olivares and C. Juárez Ramı́rez

During the characterization of sediments formed in pipelines transporting

hydrocarbons, the knowledge of the microbiological diversity becomes very
interesting, especially when it is related to microbiologically influenced
corrosion (MIC). The presence of microorganisms is considered as one of the
factors that affect the corrosion processes occurring at the pipeline; therefore,
their corrosiveness must be determined. In this way, the identification of new
species affecting the MIC processes is still considered relevant. In this work, the
effect of Clostridium celerecrescens upon the corrosion of API KL 52 steel was
evaluated. This microorganism was isolated and identified from the sediments
collected during the inner cleaning procedures of a gas pipeline. The polarization
resistance (PR) and electrochemical impedance spectroscopy (EIS) techniques
were considered to estimate the microorganism behavior during the corrosion
process. The results were complemented with a metal surface analysis, using a
scanning electron microscope (SEM). The resistance values induced by the
presence of the microorganisms clearly indicated that C. celerecrescens has an
effect on the corrosion process occurring at the API XL 52 steel surface.

1 Introduction the inner cleaning procedures of a gas pipeline. The polarization

Microbiologically influenced corrosion (MIC) has long been
studied for many years by the scientific community, and different
areas have contributed to the understanding of this subject.
Because of its economical importance, special interest has been
given to microorganisms growth occurring at installations related
to the oil industry. Nowadays, there are many publications
concerning the study of the corrosion processes caused by the
activity of sulfate reducing bacteria (SRB) in wells, pipelines, and
storage tanks. Nevertheless, the identification of other micro-
organisms and their effect on the corrosion processes is still
considered of particular interest.
In this work, the effect of Clostridium celerecrescens upon the
corrosion of API KL 52 steel was evaluated. This microorganism
was isolated and identified from the sediments collected during
O. A. Ramos Monroy, N. Ruiz Ordaz, C. Juárez Ramı́rez
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,
Prolongación de Carpio y Plan de Ayala, México D. F., C. P. 11340
(México)
M. J. Hernández Gayosso, G. Zavala Olivares
Instituto Mexicano del Petróleo, Grupo Corrosión. Eje Central Lázaro
Cárdenas 152, Col. San Bartolo Atepehuacan, México D. F., C. P. 07730
(México)
E-mail: mjazmin@imp.mx
resistance (PR) and electrochemical impedance spectroscopy
(EIS) techniques were considered to estimate the microorganism
behavior during the corrosion process. The results were
complemented with a metal surface analysis, using a scanning
electron microscope (SEM).
2 Methodology
2.1 Microorganisms isolation
The microorganisms considered in this work were isolated from
the sediments built up upon the corrosion coupons installed in a
gas pipeline located in the marine region of Mexico. The products
were incorporated on site to the PBS medium [1] (NaCl: 8.7 g/
L, KH2PO4: 0.4 g/L, K2HPO4: 1.23 g/L, ascorbic acid diluted to
5%: 20 mL/L, and resarzurine diluted to 1%: 1 mL/L; pH: 7.0),
considering anerobic conditions. After that, the samples were
sent to the laboratory and the microorganisms were maintained
in Postgate C medium (Table 1) [2].
2.2 Identification of the bacterial species
The identification of the bacterial species was carried out through
the amplification of a DNA specific region, by means of the
polymerase chain reaction (PCR) [3].

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Materials and Corrosion 2011, 62, No. 9
Table 1. Composition of Postgate C medium
Compound Concentration (g/L)
KH2PO4 0.5
NH4Cl 1.0
Na2SO4 4.5
MgSO4  7H2O 0.06
Sodium lactate 60% (p/v) 6.0
CaCl2  2H2O 0.06
FeSO4  7H2O 0.004
Yeast extract 1.0
Sodium citrate 0.3
NaCl 15
Resarzurine 0.5
The PCR was obtained using an Applied Biosystems
GeneAmp PCR System 2400 equipment, considering the
following program: (I) denaturalization at 94 8C during 5 min,
(II) 35 cycles of 1 min at 94 8C, (III) alignment at 55 8C for 1 min,
(IV) extension at 72 8C for 2.5 min, and (V) 10 min for the final
extension at 72 8C. Once the amplification was done, the samples
were cooled for 10 min at 4 8C.
For the identification of microorganisms, the following
initiators were used: (8FLP 50 -GCG GAT CCG CGG CCG CTG
CAG AGT TTG ATC CTG GCT CAG-30 ) Forward and 13B (50 -
CGG GAT CCC AGG CCC GGG AAC GTA TTC AC-30 ) Reverse
[4]. These initiators can produce amplification products of
approximately 1400 pb of 16S rDNA. The sequences obtained
were processed in the data base of the National Center for
Biotechnology Information (NCBI): http://www.ncbi.nlm.nih.-
gov, using the Basic Local Alignment Search Tool Program
(BLAST1) [5].
2.3 Kinetics growth
The microorganisms culture was inoculated in vials with 100 mL
Postgate C medium. API XL 52 steel coupons (polished and
degreased [6]) were placed into the vials, which were incubated at
room temperature. These vials were used as ‘‘sacrificial’’ to
determine the consortium kinetics growth, following the most
probable number (MPN) method [1, 7]. The planktonic and
sessile microorganisms growth was determined separately.
For planktonic microorganisms, 1 mL of culture was taken
directed from the bulk medium, and then diluted with 3 series of
10 vials each.
For sessile microorganisms, the steel coupon was placed into
a vial containing 10 mL of saline solution; then ultrasonic
cleaning bath equipment was used to remove the bacteria from
the metal surface; 1 mL was taken from this solution and then
diluted with 3 series of 10 vials each.
2.4 Kinetics growth determination using
electrochemical techniques
To determine the effect of the microorganisms upon the steel
corrosion rate, PR and EIS techniques were considered. The
experiments were carried out in a 1 L standard cell, with a three
electrodes system: API XL 52 steel as working electrode, a
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Corrosion of API XL 52 steel 879
graphite rod as auxiliary, and a saturated calomel electrode as
reference. The electrolyte used was Postgate C medium. Nitrogen
gas was bubbled to remove all the oxygen and to maintain
anerobic conditions. The measurements were obtained with a
Solartron SI 1280B potentiostat, and a PC was used for data
recording. One test was carried out every 24 h, for 30 days. The
steel corrosion effect when exposed to the Postgate C medium
was determined under the sterile conditions (reference system)
and in the presence of microorganisms.
2.4.1 Polarization resistance
A potentiodynamic method was used to obtain the current–
potential relation, applying W10 mV overpotential with respect to
the free corrosion potential, and considering a sweep of 10 mV/
min. CorrViewTM software was used for data management.
2.4.2 Electrochemical impedance spectroscopy
The measurements were done under the following conditions:
W10 mV signal amplitude and a frequency range of 1000–0.1 Hz,
recording 61 points per test. The data were analyzed with Zview1
software.
2.5 Surface analysis
Once the electrochemical experiments were done, the working
electrodes were placed into a 2.5% glutaraldehyde solution for 4 h
at 4 8C. After that, 1% osmium oxide was used for 2 h at 4 8C. The
samples were then dehydrated with four ethanol solutions (25, 50,
75, and 100%). To eliminate the ethanol from the metal surface,
the coupons were dried at their critical point with CO2 (CO2
liquefying point, temperature:
120 8C, pressure: 1800 psi) for
40 min. The coupons were covered with a thin gold film before
surface analysis at the SEM. With this preparation, microorgan-
isms can be observed at the metal surface. However, to analyze
the damage caused by the corrosion processes at the metal
surface, the biofilm and the corrosion products were removed
from the coupons and a direct surface observation was carried out
with the SEM [8].
3 Results and discussion
According to the 16S rDNA gene amplification, the microorgan-
isms isolated from the corrosion coupons have a 99% similarity to
C. celerecrescens. This species has been identified previously in the
gas pipeline, along with some SRB, such as Desulfovibrio
vietnamensis and Clostridium sporogenes [9].
C. celerecrescens is strict anerobic species and no evaluation
has been done to know its effect on the steel corrosion process.
3.1 Polarization resistance
The measurements obtained from the PR technique (Fig. 1)
indicated that the API XL 53 steel, exposed to the Postgate C
medium without microorganisms, exhibited low resistance
polarization values during the first 48 h, followed by an increase
and above 50 000 V cm2 could be observed at the end of the
experiment. These results indicate that at the beginning the bare
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
880 Monroy, Gayosso, Ordaz, Olivares and Ramı́rez
Figure 1. Resistance polarization for API XL 52 steel exposed to Postgate C medium, under sterile conditions and in the presence of C. celerecrescens
metal surface was under a high corrosion process, which
decreased with time.
During the first 350 h, the steel exposed to Postgate C
medium and in the presence of C. celerecrescens exhibited a similar
behavior to that observed at sterile conditions. However, after that
time the resistance polarization values decreased, indicating that
the corrosion processes become more important, probably caused
by effect of the biofilm developed at the working electrode
surface.
3.2 Electrochemical impedance spectroscopy
The metal samples exposed to the Postgate C medium, under
sterile conditions and in presence of C. celerecrescens were
individually analyzed with the EIS technique. The initial Nyquist
diagram obtained for the reference system (Fig. 2a) indicated a
Figure 2. EIS results obtained for API XL 52 steel exposed to Postgate C medium, under sterile conditions: (a) Nyquist diagrams, (b) equivalent
circuit, and (c) resistance values with time
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Materials and Corrosion 2011, 62, No. 9
depressed semicircle, with resistance values around 900 V cm2.
The diagrams obtained at 284 and 792 h exhibited bigger
diameters, which indicated an increase with time of the resistance
value. Under these conditions, it is established that the corrosion
process occurred during the first experimental hours, and then
lower corrosion rate was observed, as the exposition time
increased.
An equivalent circuit was proposed using the Zview program
(Fig. 2b). With this, a schematic representation of what is
happening at the metal surface may be considered. The following
elements were considered for the equivalent circuit:
1. A resistance Rs, considered as solution resistance.
2. A parallel combination of a resistance, Rct, which represents
the charge transfer resistance and a constant phase element,
CPE, considered as the double layer capacitance.
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Materials and Corrosion 2011, 62, No. 9 Corrosion of API XL 52 steel 881

Figure 3. EIS results obtained for API XL 52 steel exposed to Postgate C medium, in the presence of C. celerecrescens: (a) Nyquist diagrams, (b)
equivalent circuit used during the first 300 h, (c) equivalent circuit used after 300 h, and (d) resistance values with time

In a general way, a CPE is used instead of a capacitor, to
compensate some deviations from the ideal behavior. In practice,
the semicircles obtained are not perfect and it is very common to
observe depressions, as shown in this figure. From data fitting,
the value for each element in the system is established. In this
case the solution resistance values were not significant, below
4 V cm2, while the initial charge transfer resistance values were
around 1000 V cm2 and a sudden increase was observed in the
next hours. According to this, the steel exposed to the sterile
medium exhibited an initial corrosion rate, which decreased with
exposition time.
For the steel exposed to the medium inoculated with
microorganisms, during the first hours Nyquist diagrams,
similar to those observed under sterile conditions, were obtained
(Fig. 3a). These curves were adjusted using the same equivalent
circuit previously proposed (Fig. 3b). However, after 300 h a
Warburg element (W0) had to be incorporated, due to a
diffusion effect observed during the experiment. When simulat-
ing this circuit, values for solution resistance, charge transfer
resistance, and resistance associated to the diffusion process were
obtained. The total resistance values obtained are shown in
Fig. 3d.

Figure 4. Kinetics growth for planktonic and sessile microorganisms in Postgate C medium

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882 Monroy, Gayosso, Ordaz, Olivares and Ramı́rez
Figure 5. SEM images obtained for API XL 52 steel: (a) before being exposed to C. celerecrescens; (b) biofilm formed at the coupon surface; (c) and
(d) localized corrosion caused by the microorganisms
Initially, the kinetics process for the steel sample exposed to
C. celerecrescens exhibited similar behavior to that obtained under
sterile conditions, but after 300 h an increment on the corrosion
rate is observed, with resistance values around 2500 V cm2 after
800 h incubation time. The resistance values induced by the
presence of the microorganisms clearly indicated that
C. celerecrescens has an effect on the corrosion process occurring
at the API XL 52 steel surface.
According to the microorganisms kinetics behavior observed
(Fig. 4), the increase on the corrosion rate agrees with the
deposition of sessile microorganisms at the metal surface, which
promotes the accumulation of aggressive metabolic products at
the steel–biofilm interface. The microorganisms population
increased notably after 200 h exposition time.
The resistance values obtained through both electrochemical
techniques are consistent and exhibited the same behavior for the
evaluated conditions.
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Materials and Corrosion 2011, 62, No. 9
3.3 Scanning electron microscopy (SEM)
Different SEM images of API XL 52 steel coupons, before and
after being exposed to C. celerecrescens for 6 weeks, are shown in
Fig. 5. According to the images, the microorganisms are immers-
ed in the biofilm that is covering the metal surface. At the same
time, a localized corrosion process, which is originated by the
microorganisms at the metal surface, is observed in the images.
4 Conclusions
The corrosion induced by C. celerecrescens seems to be low,
compared to that reported for D. vietnamensis, which was isolated
from the same gas pipeline [9]. However, the presence of
C. celerecrescens should be considered as one of the factors
affecting the corrosion processes occurring at the inner side of the
gas pipeline.
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Materials and Corrosion 2011, 62, No. 9
The works related to the identification of bacteria associated
to microbiologically induced corrosion, allow extending the
understanding of the microbial species developed at pipelines
transporting hydrocarbons. At the same time, their individual
effect on the corrosion processes can be established. Although the
corrosion rate is regularly monitored using either weight loss
coupons or electrical resistance probes, the results obtained
include many variables that affect the process and it is very
difficult to establish which of them has bigger influence.
5 References
[1] NACE TM0194-2004, Field Monitoring of Bacterial Growth in
Oilfield Systems, National Association of Corrosion Engin-
eers, Houston, 2004.
[2] J. R. Postgate, The sulphate reducing bacteria, 2nd edition,
Cambridge University Press, New York 1984, p. 32.
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Corrosion of API XL 52 steel 883
[3] H. P. Vosberg, Hum. Genet. 1989, 83, 1.
[4] D. A. Relman, in: H. D. Persing, F. T. Smith, C. F. Tenover, S.
T. White (Eds.), Diagnostic Molecular Microbiology. Principles
and Applications, ASM Press, Washington, DC, USA, 1993.
[5] S. F. Altschuld, W. Gish, W. Miller, W. Myer, J. Mol. Biol. 1992,
215, 403.
[6] NACE RP0775-2005, Preparation, Installation, Analysis, and
Interpretation of Corrosion Coupons in Oilfield Operations,
National Association of Corrosion Engineers, Houston,
2005.
[7] R. Blodgett, Bacteriological Analytical Manual, Appendix 2.
Most Probable Number from Serial Dilutions. U.S. Food and
Drug Administration, 2003.
[8] J. J. Bozzola, D. L. Russell, Electron Microscopy: Principles and
Techniques for Biologists. 2nd edition, Jones and Bartlett Press,
New York, USA, 1999.
[9] M. J. Hernández-Gayosso, G. Zavala-Olivares, N. Ruiz-Ordaz,
R. Garcı́a, Mater. Corros. 2005, 56, 624.
(Received: November 19, 2009) W5582
(Accepted: December 19, 2009)
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