624 Hernández Gayosso, Zavala Olivares, Ruiz Ordaz and Garcı́a Esquivel Materials and Corrosion 2005, 56,
and Corrosion 2005, 56, No. 9
Evaluation of a biocide effect upon
microbiologically influenced corrosion of mild
steel
The microbiologically influenced corrosion (MIC) is a very dan-
gerous process, which affects the oil industry. The activity and mi-
croorganisms’ growth at the pipelines steel cause surface modifica-
tions, which can induce a more complex corrosion process. The bio-
cide evaluation for the MIC decrease has been normally based upon
microbiological tests, and just a few references mention alternating
methods which can be used as criteria for their evaluation. In this
work, a commercial biocide was tested, using different electroche-
mical laboratory techniques, to determine its effect upon a biofilm
generating bacteria consortium.
1 Introduction
The microbiologically influenced corrosion (MIC) is a very
dangerous process, which affects the oil industry, particularly
during the hydrocarbon extraction, transport and storage [1 –
3]. The activity and microorganisms growth at the pipelines
steel cause surface modifications, which can induce a more
complex corrosion process. In general, the study of the
MIC and the biocide evaluation for corrosion decreasing,
have been normally based upon microbiological tests and
gravimetric techniques, and just a few references mention al-
ternating methods, which can be used as criteria for their eva-
luation.
At the same time, most of the research on MIC has been
done considering the use of isolated strain bacteria. However,
in natural conditions the microorganisms are found in consor-
tia, and the corrosion processes could be different. Moreover,
and according to the measurements taken in the laboratory,
different strains in the system could lead to different biocide
behaviour and a specific chemical compound may have dif-
ferent responses to those expected with an isolated strain.
* M.J. Hernández Gayosso, G. Zavala Olivares,
R. Garcı́a Esquivel
Instituto Mexicano del Petróleo, Grupo de Corrosión.
Eje Central Lázaro Cárdenas 152, Col. San Bartolo Atepehuacan,
México, D.F, C.P. 07730 (Mexico),
E-mail: mjazmin@imp.mx
N. Ruiz Ordaz
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico
Nacional.
Prolongación de Carpio y Plan de Ayala, México, D.F, C.P. 11340
(Mexico)
DOI: 10.1002/maco.200503867
M. J. Hernández Gayosso*, G. Zavala Olivares,
N. Ruiz Ordaz and R. Garcı́a Esquivel
Using microbiological techniques, the biocide lethal concentra-
tion was determined, and a concentration of 200 ppm was used to
kill completely the consortium population in both, plancktonic and
sessile parts. The electrochemical techniques: Polarisation Resist-
ance (PR) and Electrochemical Impedance Spectroscopy (EIS), al-
lowed describing the corrosion process associated to the microbial
consortium and the biocide effect upon it.
The use of electrochemical techniques for the evaluation of
MIC, as well as the biocide selection and evaluation, have
been recently studied [4 – 8] and this is an interesting area
which requires more research, in order to implement and stan-
dardize laboratory experiments and field application.
The polarisation resistance (PR) is the most common elec-
trochemical technique used to evaluate corrosion rates on me-
tal surfaces. This evaluation allows measuring instantaneous
corrosion rates, and its monitoring can be continuous. There-
fore, the technique could be useful when detecting corrosion
rate changes [4, 9].
Besides the PR, there are other electrochemical techniques,
such as the Electrochemical Impedance Spectroscopy (EIS),
which also can be used to obtain information and explain the
corrosion processes occurring on the metal surface.
The EIS technique, apart from the corrosion rate values,
could provide information about the controlling corrosion
process and film formation on the metal surface. The tech-
nique also allows calculation of the system components,
such as the solution resistance, charge transfer resistance
and double layer capacitance, by means of their electric repre-
sentation in an equivalent circuit [4, 10].
2 Experimental
To evaluate the biocide effect upon biofilm generating bac-
teria, using the electrochemical techniques, PR and EIS, the
following activities were carried out:
2.1 Bacterial culture
The bacterial culture used to evaluate the biocide was iso-
lated from a residual products sample, taken from a gas pipe-
F 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Materials and Corrosion 2005, 56, No. 9
Table 1. Composition of the Postgate C medium14
Compound g/l
KH2PO4 0.5
NH4Cl 1.0
Na2SO4 4.5
MgSO4  7H2O 0.06
Sodium lactate 6.0
CaCl2  2H2O 0.06
FeSO4  7H2O 0.004
Yeast extract 1.0
Na citrate 0.3
NaCl 15
line located in the Marine Region of México, during the inner
cleaning procedure.
The microorganism’s diversity was determined using the
Thermal Gradient Gel Electrophoresis Technique (TGGE),
with amplified fragments of 16S rDNA gene. The chain reac-
tion (PCR) was carried out using the U968-GC and 1401 in-
itiators [11]. The DNA fragments generated were observed
using poliacrylamide gel electrophoresis [12]. The bacterial
group present on the surface was identified by means of
the complete gene amplification (1502 bp). Using the 8FPL
and 1492 RPL initiators [13], the obtained sequence was pro-
cessed using the National Center of Biotechnology Informa-
tion (NCBI) data base: http://www.ncbi.nlm.nih.gov.
2.2 Biocide lethal concentration
The culture was inoculated in 100 ml vials containing Post-
gate C [14] medium (Tab. 1) and API XL52 steel coupons (po-
lished and degreased). The coupons were suspended into the
solution. The consortium was incubated for about 500 h be-
fore the biocide addition. The biocide used in the experiments
was Glutaraldehyde, added at 100 and 200 ppm separately.
24 h after the biocide was added, the Most Probable Number
(MPN) was determined for sessile and plancktonic microor-
ganisms.
2.2.1 Plancktonic microorganisms
1 ml of the previous solution (culture þ Postgate
C þ biocide) was inoculated in vials containing 9 ml of Post-
gate C medium, to determine the MPN of plancktonic micro-
organisms.
2.2.2 Sessile microorganisms
The suspended coupons, exposed to the biocide action,
were removed from the initial solution and placed in vials con-
taining 10 ml of saline solution and solid-glass beads, in order
to remove the bacteria from the metal surface when shaking
the vial; 1 ml from this solution was used for the MPN method
2.3 Corrosion rate determination using electrochemical
techniques
These experiments were carried out in a 1 l standard cell,
with a three electrode system: API XL52 steel as working
Biocide effect on MIC 625
electrode, a graphite rod as auxiliary and a saturated calomel
electrode (SCE) as reference. The electrolyte used was 800 ml
Postgate C medium. Nitrogen gas was bubbled to remove all
the oxygen and maintain anaerobic conditions. The electro-
chemical cell was connected to an ACM 772 potentiostat,
and a PC was used for data recording. All experiments lasted
approximately 1 month, and during this time, several PR and
EIS tests were carried out.
To evaluate the biocide effect upon the MIC using electro-
chemical techniques, 500 h after the experiment was started, it
was considered that a biofilm was formed at the steel surface
and the biocide was added to the cell.
2.3.1 Polarisation Resistance technique
A potentiodynamic method was used to obtain the poten-
tial-current (E-I) ratio, applying a
10 mV overpotential,
with respect to the free corrosion potential, Ecorr. The Se-
quencerÒ and Core runningÒ software programs were used
for data management, along with the V4 AnalysisÒ software
for data analysis.
From this technique, resistance values are obtained (Rp),
which are used to calculate the corrosion current density, ac-
cording to:
b
icorr ¼ ð1Þ
Rp
Where, b is the Tafel slope, and a value of 0.026 Volts dec-
ade1 was considered.
Faraday’s Law can be used to relate the corrosion current
density to the corrosion rate, with the next equation:
icorr
corrosion rate; CR ¼ K EW ð2Þ
d
Where: K ¼ constant, EW ¼ equivalent weight and
d ¼ density
2.3.2 Electrochemical Impedance Spectroscopy technique
These experiments were carried out considering a fre-
quency range of 1000  0.1 Hz, and
10 mV signal ampli-
tude, recording approximately 40 points per test. Data record-
ing was made using the SequencerÒ and Core runningÒ soft-
ware programs, along with the ZviewÒ software for data ana-
lysis.
Using the ZviewÒ software, the system behavior was simu-
lated and adjusted to a proposed equivalent circuit, consider-
ing the characteristics of the corrosion process and the resist-
ance value for each proposed element was calculated. To eval-
uate the corrosion rate, the charge transfer resistance and the
resistance induced by the biofilm were considered and used in
equations (1) and (2).
3 Results and discussion
Using the TGGE technique, it was found that the consor-
tium used in this work is constituted by 5 different bacteria
species, as shown in Fig. 1. These 5 bacteria are observed
in the aqueous medium (plancktonic microorganisms), and
626 Hernández Gayosso, Zavala Olivares, Ruiz Ordaz and Garcı́a Esquivel
Fig. 1. Pattern band for amplified fragments of 16S rDNA, ob-
tained from TGGE Technique
only one is observed at the metal surface (sessile microorgan-
ism). The pattern band for the sessile microorganism is ob-
served as well for plancktonic bacteria.
The 16S rDNA gene of the sessile microorganism is formed
of approximately 1500 bp and according to the gene amplifi-
cation there is a similarity of 97% with Desulfovibrio vietna-
mensis. This species has been isolated previously in the oil
industry [15]. It is very important to point out that the Desul-
fovibrio vietnamensis is the species at the metal surface and
the main responsible for MIC processes, by means of the me-
tabolic products formed at the interface. However, the planck-
Table 2. Biocide effect upon microorganisms
No biocide conc. 100 ppm biocide conc.
Plancktonic bac/ml Sessile Bac/cm2 Plancktonic bac/ml
4.3  103 4.3  106 9.3  101
Microorganisms killed 97.8%
Materials and Corrosion 2005, 56, No. 9
tonic microorganisms should have some influence on the cor-
rosion process.
According to the results shown in Tab. 2, the biocide exhib-
ited good performance for both, sessile and plancktonic mi-
croorganisms, indicating that this compound was able to pe-
netrate the biofilm formed by the consortium.
These results are in good agreement with those reported by
Eagar et al. (1988) [16] and Grab and Alan (1992) [17], where
biocide concentrations between 100 and 200 ppm are reported
to be sufficient to eliminate sessile and plancktonic microor-
ganisms.
In this study, the biocide effect was very similar for both,
plancktonic and sessile microorganisms, probably due to the
high biocide concentration. However, at lower concentrations
better biocide performance for plancktonic than for sessile mi-
croorganisms is expected, as indicated in previous studies
[18 – 20]. Therefore, the experiments were carried out using
200 ppm biocide concentration.
3.1 Polarisation Resistance technique
The corrosion rate values obtained when using the Polari-
sation Resistance technique, in presence of the microbial con-
sortium and after the biocide was added are shown in Fig. 2.
From this graph, it is observed that under sterile conditions,
the steel corrosion rate is very low, and values around
0.02 mm/y were induced by the microbial consortium. After
200 h of bacteria action during the experiments, the corrosion
rate gradually increased until values about 0.5 mm/y at ap-
proximately 500 h. Later on, the biocide was added to the me-
dium, decreasing the corrosion rate. This situation makes evi-
dent the biocide effect upon the microorganisms and the cor-
rosion rate observed.
200 ppm biocide conc.
Sessile Bac/cm2 Plancktonic bac/ml Sessile Bac/cm2
1.5  102 9  100 7.5  101
96.5% 99.8% 98.2%
Fig. 2. Corrosion rate values obtained
with the Polarisation Resistance tech-
nique, in presence of the microbial
consortium and after the biocide was
added
Materials and Corrosion 2005, 56, No. 9
Fig. 3. Nyquist diagrams obtained for API XL 52 exposed to the bacterial culture. a) 1 to 300 h exposition time, b) 300 to 500 h exposition
time, c) Diagrams obtained after biocide addition, 500 to 800 h exposition time
3.2 Electrochemical Impedance Spectroscopy technique
The Nyquist diagrams, obtained before and after biocide
addition, are shown in Fig. 3. According to this Figure, during
the first 300 h (Fig. 3a), semicircle curves were observed, and
an activation control process was assumed to be occurring at
the metal surface. After that time, a diffusion control process
was observed, indicated by straight lines in the Nyquist dia-
grams (Fig. 3b). The reason for this behavior could be an in-
crement in the biofilm thickness.
The Nyquist diagrams obtained from the beginning up to
300 h experimental time were adjusted using the equivalent
circuit I, shown in Fig. 4. The curves obtained after that
time were adjusted using the equivalent circuit II. For all cur-
ves, the Zview program, used to obtain the equivalent circuits,
indicated errors below 10%.
According to the results, it seems that the microorganisms
grow at the metal surface from the beginning of the experi-
ments, and this situation is indicated in the equivalent circuits
by the R1 and CPE2 elements, which represent the resistance
and capacitance induced by the biofilm. At the same time a
diffusion process was detected during the experiments after
300 h exposition time.
It is interesting to point out that the diffusion process re-
mains even after the biocide addition. This indicates that
although the microorganisms are killed by the biocide action,
the glutaraldehyde does not affect the biofilm structure at the
metal surface.
Once the real curves were fitted to the equivalent circuits
proposed, the capacitance values for CPE1, CPE2 and W1 at
Fig. 4. Equivalent circuits used to adjust the Nyquist diagrams
Biocide effect on MIC 627
different conditions were calculated and are shown in Fig. 5.
During the first 500 h, the values increase, indicating a very
clear influence upon the capacitance induced by the surface
biofilm (CPE2).
The bacterial population growth at the metal surface, as
well as the production and accumulation of exopolysacchar-
ide, metabolites and corrosion products, induced an increment
in the biofilm thickness, and make it more compact with time,
originating higher capacitance values and promoting a diffu-
sion control process, which was clearly observed after 300 h
experimental time.
After 500 h experimental time, when the biocide is added to
the system, the capacitance induced by the biofilm (CPE2)
remains constant. This situation could be explained consider-
ing that, although the biofilm still is adhered to the metal sur-
face, the microbial activity has stopped.
Fig. 6 shows the corrosion rate values obtained with the im-
pedance technique, for the metal exposed to the bacterial cul-
ture, before and after the biocide addition. Initial corrosion
rate values below 0.05 mm/y were observed. As observed
when using the PR technique, after 200 h there was a gradual
increment on the corrosion rate, up to around 0.3 mm/y at
500 h. When the biocide was added into the medium, the cor-
rosion rate decreased in a similar way to that presented by the
PR technique.
The corrosion rate values observed at sterile conditions
were very low (below 0.02 mm/y), even when the biocide
was added to the system. This situation indicates that the glu-
taraldehyde itself does not have any effect on the metal corro-
sion rate.
The results obtained with both techniques, RP and EIS, ex-
hibit a narrow correlation in the system corrosion process.
Both techniques allow detecting corrosion rate changes, as re-
sult of microorganisms’ presence and the biocide effect upon
them.
When the biocide is incorporated to the system, a decre-
ment on the corrosion rate is observed, probably because
the microorganisms are killed by the biocide action and the
production of hydrogenase enzyme is stopped, avoiding the
cathodic depolarization at the metal surface.
However, in spite of the lethal biocide effect upon the mi-
croorganisms, a biofilm effect upon the metal surface can still
be observed, and the corrosion rate measured after the biocide
addition could be due to the presence of aggressive metabo-
lites at the interface, as well as to the presence of iron sulfide,
which is one of the corrosion products accumulated at the me-
628 Hernández Gayosso, Zavala Olivares, Ruiz Ordaz and Garcı́a Esquivel
tal surface. The iron sulfide induces a galvanic effect, as this
compound is cathodic to the steel.
A reduction on the corrosion rate induced by microorgan-
isms to similar values to those observed at sterile conditions,
only could be achieved if the biofilm is removed from the me-
tal surface, along with the metabolic products. However, un-
der the conditions considered in this work (batch culture), the
biofilm removal allows that the aggressive metabolites were
incorporated into the electrolyte and still affecting the metal
surface, promoting higher corrosion rate than for sterile con-
ditions. Considering this situation, a continuous (loop) culture
is suggested.
The results obtained with the techniques PR and EIS, make
evident the microbial consortium effect on the increment of
corrosion rates for this system. At the same time, the biocide
effect can be observed when, after the chemical addition, the
steel corrosion rate decreased. Taking this information into
account, both techniques could be considered to determine
the biocide influence upon the microbiologically influenced
corrosion.
Materials and Corrosion 2005, 56, No. 9
Fig. 5. Capacitance values for the
equivalent circuit used to adjust
the curves obtained for the metal
exposed to the bacterial culture, be-
fore and after biocide addition
Fig. 6. Corrosion rates ob-
tained with the Electrochemical
Impedance Spectroscopy tech-
nique, in presence of the micro-
bial consortium and after the
biocide addition
4 Conclusions
Based on the results obtained in this work, it is observed
that:
The bacterial culture isolated from the Xicalango-Atasta
gas pipeline induces an increment in the corrosion rate of
the API XL52 steel, possibly through the following mechan-
isms: 1) cathodic depolarization, 2) acidic metabolites con-
centration at the metal surface and 3) corrosion products ac-
cumulation (iron sulfide).
The Electrochemical Techniques: PR and EIS, exhibited
good correlation when evaluating the corrosion kinetics of
the systems considered in this work. Using both techniques,
it is possible to determine the corrosion rate. Additionally, the
EIS technique allows to establish data related to the mechan-
istic variables of the process.
The lethal effect of the glutaraldehyde upon the bacterial
culture could be observed through the experimental collected
data. Both techniques were sensitive enough to detect a de-
crease in the steel corrosion rate, as a result of biocide addi-
tion.
Materials and Corrosion 2005, 56, No. 9
Furthermore, some biofilm characteristics could be deter-
mined with the equivalent circuits proposed to simulate the
corrosion processes observed in this work.
According to the results, both techniques: PR and EIS, were
found to be suitable for describing de corrosion process induc-
ed by microorganisms, as well as the biocide effect upon the
system.
5 Acknowledgments
The authors wish to thank the Instituto Mexicano del Pet-
róleo (IMP), Escuela Nacional de Ciencias Biológicas
(ENCB) and Consejo Nacional de Ciencia y Tecnologı́a
(CONACyT), for their support in the production of this work.
6 References
[1] B. J. Little, P. A. Wagner, F. Mansfeld, Microbiologically In-
fluenced Corrosion. NACE International, Houston TX, 1997.
[2] D. H. Pope, E. Morris III., Materials Performance 1995, 23.
[3] R. Towers, Protective Coatings Europe 2000, 30.
[4] G. H. Canullo-Vunk, J. C. Steelhammer, J. Lukanich, Proc.-
Int. Water Conf. 1995, 56, 432.
[5] G. A. Sussex, Proc. Corros. Prev. 1995, 9, 10.
[6] G. Salvago, G. Fumagalli, P. Cristiani, G. Rocchini, European
Federation of Corrosion Publications 1995, 15, 301.
[7] M. F. De Mele, Trends Corrosion Ressearh. 1997, 2, 59.
Biocide effect on MIC 629
[8] J. Alhajji, M. Valliapan, Proc. World Pet. Congr. 15th 1998, 2,
847.
[9] F. Mansfeld, B. Little, Corrosion Science 1991, 32, 247.
[10] L. L. Shreir, R. A. Jarman, G. T. Burstein, Corrosion, metal
and environment reactions. Ed. Butterworth Heinemann LTD.
Vol 1 3a edition 1995.
[11] A. Felske, B. Engelen, U. Nübel, H. Backhaus, Appl. Environ.
Microbiol. 1996, 62, 4162.
[12] C. A. Eichner, R. W. Erb, K. Timmis, I. Wagner-Dobler, Appl.
Environ. Microbiol. 1999, 65, 102.
[13] A. D. Relman, Diagnostic Molecular Microbiology, 490.
American Society for Microbiology, Washington DC, 1993.
[14] J. R. Postgate, The Sulphate Reducing Bacteria, Cambridge
University Press, 1984, 32.
[15] N. D. Phuong, C. H. D. Thi, H. L. Thuy, H. Stand Lotter, An-
aerobe 1996, 2, 385.
[16] R. G. Eagar, J. Leder, J. P. Stanley, A. B. Theis, CORROSION/
88, NACE 1988, paper no. 84.
[17] L. A. Grab, B. Alan, CORROSION/92, NACE 1992, paper no.
184.
[18] J. W. Costerton, E. S. Lashen, Influence of biofilm on efficacy
of biocides on corrosion-causing bacteria. National Associa-
tion of Corrosion Engineers, USA, NACE 1984, 13 – 17.
[19] C. W. Keevil, C. W. Mackerness, International Biodeteriora-
tion 1990, 26, 169.
[20] H. A. Videla, Manual of Biocorrosion. Eds. CRC Lewis Pub-
lishers. USA. 1996.
(Received: March 24, 2005) W 3867
(Final version: May 6, 2005)