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Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623

  Sulfur cycle of microbial corrosion on carbon steel in soil model

                                Mataqi, K.Y. *, Akbar, B.H.**
              *(Department of Biotechnology, Kuwait Institute for Scientific Research, Kuwait)
              ** (Department of Biotechnology Kuwait Institute for Scientific Research, Kuwait)



ABSTRACT                                                which either oxidize or reduce sulphur compounds as
         This study examined the effects of             some part of their life process. Although many
Desulfovibrio desulfricans and bacteria consortia       culturable bacterial types with known corrosion
on the rate of carbon steel corrosion in soil model.    effects have been identified [5], in both aquatic and
Microbial corrosion was measured using the              terrestrial environments the primary corrosion-
corroded mean depth after 56 days incubation            causing bacteria are the sulfate-reducing iron-
under aerobic and anaerobic conditions. The             oxidizing bacteria. Desulfovibrio desulfricans is
effects of water content and dissolved oxygen in        examined in this report as a source of Sulfate
soil on the corrosion rate were also analyzed.          reducers, which is found to exist in all soil and water
Results showed that aerobic conditions increased        types, as well as lives symbiotically with facultative
corrosion rate. Moreover, sole Desulfovibrio            anaerobic bacteria [6]. Sulfate-reducing bacteria
desulfricans treatment ceased the corrosion as a        (SRB) are a group of anaerobic diverse organisms in
protective ferrous sulfide film formed on the           which have varied morphological and nutritional
carbon steel. While the heterogeneous biofilm of        characteristics. They utilize organic matter to produce
the bacterial consortia formed uneven oxygen            sulfide by either reducing or oxidizing sulfate
concentration which accelerated the corrosion.          compounds [7] , as a source of energy. Therefore,
                                                        sulphate (SO42–) can be reduced to sulphide (S2–) by
Keywords - Bacteria , Carbon steel, Corrosion,          SRB leading to the generation of hydrogen sulfide as
SRB, Desulfovibrio desulfricans                         a metabolic bi-product. Both physical and chemical
                                                        processes transfer Hydrogen sulfide (H2S) across the
                I. Introduction                         air and water boundaries to environments where
Corrosion is an ever-present degradation mechanism      chemoautotrophic bacteria oxidize the sulfide to
in wetted components and systems. There are many        sulfuric acid [8]. The corrosion process will hence
forms of corrosion in metals, that include; pitting,    occur by the reaction of the biogenic sulfuric acid
stress corrosion, general corrosion, galvanic           with the metallic surfaces [9]. In order to evaluate the
corrosion, and others [1], in which microbiologist      significance of microbial corrosions, a look at the
have recognized and are constantly addressing.          economical perspective on such effects is essential.
When a system ` first encounters microbial corrosion    In 2001, the cost of microbial-influenced corrosion
(MC) such event usually occurs during the system        on oil and gas industries accounted for about $2
initial exposure to an aqueous environment, such as     billion annually [10]. Microbial induced corrosion in
during hydrotest, wet lay-up, or moist soil. The        the US economy has reached $350 billion annually as
presence of certain bacteria in an environment will     of 2010 [11]. Mild steel in used widely in piping
lead to the production of microbial corrosions. This    systems, storage tanks, cooling towers and aquatic
mode of corrosion can be accelerated by microbial       structures and is the most readily corroded metals
organisms, either because they manufacture              [12]. Since bacteria form colonies beneath which
aggressive species, such as protons or sulphide ions,   corrosion can occur, prevention of colonization is one
or because they catalyze the electrochemical            of the potential ways to prevent MC. This report will
reactions themselves [2]. MC induction also requires    examine the effects of Desulfovibrio desulfricans and
the presence of nutrients and water to ensure the       bacteria consortia on the rate of carbon steel
survival and growth of microorganisms. Thus, the        corrosion in soil model with respect to dissolved
ability of microorganisms to sense and rapidly          oxygen and water content distribution in such a
response to harsh environmental changes is vital for    system.
their survival and MC capabilities [3]. Microbes can
grow in fluids with pH values ranging from -1 to 10,             II. Materials and Methods
where -1 is the most acidic concentration, and derive   2.1 Organisms
energy from organic or inorganic materials.                      The sulfate reducing bacterial stain
Moreover, microbes can survive temperatures which       Desulfovibrio desulfricans DSMZ 642 was used as
range from -4 to 210oF (-20 to 99oC) [4]. The           the model SRB in this study. Activated sludge from a
majority of the active organisms involved in            municipal wastewater plant was used as model
corrosion are bacteria, about 1-5 micrometers long,     Bacteria consortia.


                                                                                                617 | P a g e
Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623
2.2 Culture medium                                       30mL vial bottle; (2) butyl rubber plug; (3) aluminum
Modified Baar`s Medium (MBM), containing (in             cap; (4) Na2S solution in 500µL microtube; (5)
g/L) anhydrous sodium citrate 5.0, 50% sodium            carbon steel coupons; (6) culture medium in SiO2
lactate 4.9, yeast extract 1.0, NH4Cl 1.0,               soil. (B) Aerobic conditions: (7) 50mL centrifuge
MgSO4.7H2O 2.0, CaSO4 1.0, and K2HPO4 0.5, was           tube; (8) silicone plug.
used as culture medium.             The pH, sulfate      To create artificial model soil, 30mL vials (Nichiden
concentration and total organic carbon concentration     Rika Garasu, Hyogo) containing 25g silicon dioxide
(TOC) of MBM are 7.0, 1500 mg/L, and 2500 mg/L,          (SiO2) sand, a 500µL microtube, and three carbon
respectively. SRB and activated sludge were pre-         steel coupons were autoclaved, sealed with a butyl
incubated in MBM at 37oC and 25oC, respectively,         rubber plug and an aluminum cap, and replaced with
for seven days. Then, one-tenth of these pre-            N2 gas (N2 >99.9995%) (Figure, 1A). To maintain
incubated culture broths were inoculated into test       the anaerobic conditions throughout the experiment,
culture medium. The inoculated SRB size was 4x105        400µL of 27.2g/L Na2S solution was poured into the
colony forming unit (CFU) CFU/mL D. desulfricans         microtube. Then, 7.5mL of the degassed culture
and 3x107 CFU/mL facultative anaerobic bacteria.         medium was inoculated. To maintain aerobic
The inoculated activated sludge size was 7500mg/L        conditions, a 50mL centrifuge tube containing 25g
mixed liquor suspended solid (MLSS). Sterile MBM         SiO2 and three carbon steel coupons was autoclaved.
was prepared by autoclaving the medium at 120̊ C for     Followed by the inoculation with 7.5mL of the
20min.                                                   culture medium (Fig, 1B).
2.3 Carbon steel coupons preparation                     2.6 Measurement of water content on corrosion
Rectangular carbon steel coupons (20x10mm and            To investigate the effects of water content on
0.35mm thick) were cut from a sheet stock. The           corrosion, 4.5 or 1.5mL culture medium was
composition of the carbon steel coupons was (in wt       inoculated. Water content (WC) was defined as the
%) 99.71 Fe, 0.03 C, 0.01 Si, 0.19 Mn, 0.013 P,          ratio of water volume to void of the model soil.
0.017 S, 0.0017 N, and 0.026Al. The surface was          Since the void of 25g SiO2 7.5mL, addition of 7.5,
wet-polished with 800-grid polishing paper. The          4.5 and 1.5mL of culture medium resulted in 100, 60
polished coupons were cleaned ultrasonically in          and 20% WC, respectively. To keep the water
acetone for 15min, weighed, air-dried, and stored in a   constant in the test tube, sterile water was added and
desecrator.                                              the tube was centrifuged (1500G, 3min) every week.
2.4 Corrosion Measurements                               After incubation for 0, 7, 14, 28, and 56 d, saline
The corroded mean depth (CMD) was used as an             (NaCl 8.0 g/L, KCl 0.2 g/L) was added to the model
indicator of the extent of corrosion. Corrosion          soil. The model soil was sonicated for 20 sec, and
products were selectively removed from tested            mixed with a spatula.
coupons by incubating at 60oC for 60 min in a 10wt%      2.7 Measurement of Total Organic Carbon and
HCl solution with 0.3vol% Ibit, a polycationic amine          Sulfate Concentrations
derivative that protects metal steel surface from HCl.   Sulfate concentration and TOC concentration in the
Then the treated coupons were weighed. Mass loss         culture was measured by sulfur analyzer Antek (9000
of the carbon steel coupon was divided by the            Series) and a TOC analyzer, respectively. The pH of
specific gravity of iron (7.86g/cm3) and by the area     the culture tenfold-diluted solution was measured by
(cm2) of the carbon steel coupon, and CMD was            a pH meter. Corrosion products were selectively
derived.                                                 removed from the corroded carbon steel coupons as
2.5 Model soil preparation                               described above. The roughness of the coupon was
                                                         analyzed by a laser 3D profile microscope (VK-
                                                         8500).
                                                         2.8 Bacterial count
                                                         To count the aerobes in the culture, saline was added
                                                         to the model soil and the supernatant was spread-
                                                         plated on Minimized Luria-Bertani (MLB) agar plate,
                                                         containing (in g/L) polypeptone 1.0, yeast extract 0.5,
                                                         NaCl 10.0, and agar 15, in triplicate.           After
                                                         incubation for 7 d at 28oC, the observed colonies
                                                         were counted as aerobes. For the counting of
                                                         anaerobes and SRB in the experimental culture, the
                                                         supernatant was added dropwise to the plates and the
                                                         MBM agar medium with 173 (in g/L)
                                                         Fe(NH4)2(SO4)2 was poured.           The plates were
                                                         incubated in sealed jars with an oxygen absorbing
Figure 1. Experimental apparatuses for preparation of    and carbon dioxide generating agent.             After
artificial model soil. (A) Anaerobic conditions: (1)     incubation for 14 d at 37oC, the observed black



                                                                                                618 | P a g e
Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623
colonies were counted as SRB and others were           Figure 2. Time Course of CMD (A), sulfate
counted as anaerobes. The concentration of bacteria    concentration (B), TOC concentration (C) , and pH
was expressed as (CFU) per mL.                         (D) under anaerobic ( , , , ) and aerobic ( , ,
                                                       ) conditions. The test was conducted in SRB culture
                III. Results                           ( , ), bacterial consortia ( , ) and in sterile
3.1 Analysis of corroded carbon steel                  conditions as a control ( , ).
         The CMDs of carbon steel coupons under
anaerobic and aerobic conditions were compared
after 56 days incubation period.        In Bacterial   Table 1. Effect of dissolved Oxygen Existence and
consortia, the CMD under aerobic conditions reached    Bacteria on Endpoint CMD (µm) after 56 days
34.1 µm, which was 28 times higher than anaerobic      Incubation
conditions with CMD measurement of only 1.2 µm.                          SRB        Bacterial       Sterile
However, the CMD for SRB-inoculation or sterile                                     consortia
conditions reached 5.42 or 5.11 µm respectively, in
which was 8.9 or 8.8 times higher than the anaerobic
conditions (Fig. 2A, Table 1). There weren’t any        Anaerobic        0.61           1.2           0.58
significant differences between SRB and sterile
control conditions, yet in Bacterial consortia a
change was noted with 2.1 and 6.7 times increase in      Aerobic         5.42          34.1           5.11
anaerobic and aerobic treatments respectively, when
compared with sterile CMD values. These
observations indicated that existence of oxygen        Upon inoculation of SRB under aerobic conditions,
causes carbon steel corrosion in model soil.           little corrosion on the steel coupons was observed
                                                       during the 14-28 days incubation period. In the
                                                       model soil analyzed, a gap separating the soil into
                                                       upper and lower parts was observed just after
                                                       incubation for 7 days. This gap seemed to influence
                                                       corrosion acceleration, as the coupons were corroded
                                                       unexpectedly at day 7. The whole surface of coupon
                                                       immersed into the SRB culture was covered with
                                                       homogeneous black corrosion products that were
                                                       easily wiped off. When the corroded coupons were
                                                       incubated with HCl, hydrogen sulfide (H2S) gas was
                                                       produced, indicating that FeS was formed. In
                                                       addition, local corrosion under black tubercles was
                                                       observed under aerobic conditions. These
                                                       observations showed that the sole existence of SRB
                                                       didn`t accelerate carbon steel corrosion, while that of
                                                       Bacterial consortia did indeed accelerate the
                                                       corrosion process.
                                                       3.2 Influence of pH, Sulfate and TOC concentrations
                                                             on bacterial growth over time
                                                       Sulfate concentration for the SRB inoculated in both
                                                       anaerobic and aerobic conditions stopped decreasing
                                                       at day 7 suggesting that the sulfate-reducing activity
                                                       of SRB was high until this day as it began to decrease
                                                       afterwards. While, the sulfate concentration for
                                                       Bacterial consortia stopped decreasing at day 14
                                                       (Fig. 2B). Incubation of carbon steel coupons with
                                                       SRB or Bacterial consortia inoculae lead to the
                                                       depletion of TOC concentration at day 7;
                                                       furthermore, heterotrophic bacteria were inactive
                                                       during the 14-56 day incubation period (Fig. 2C) and
                                                       the concentration of aerobes decreased as well (Fig.
                                                       3A). Under anaerobic conditions, the TOC was not
                                                       consumed completely, because oxygen as electron
                                                       acceptor was limited. TOC concentration decreased
                                                       even under sterile conditions. In another experiment
                                                       with sterile conditions that did not contain carbon



                                                                                              619 | P a g e
Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623
steel coupons, the TOC concentration however did
not decrease (data not shown). This indicates that the
corrosion products adsorbed the organic carbon.
Under anaerobic conditions, SRB concentration was
decreased and became negligible during the 28-56
day incubation period (Fig. 3C). This is due to the
fact that insufficient sulfate and hydrogen sulfide that
filled the tested bottle inhibited the growth of SRB.
Under aerobic conditions, the presence of SRB
suggested the formation of anaerobic regions in the
soil.
When examining the pH concentration under
anaerobic conditions, it increased to 7.9 after
incubation for 7 days and remained constant for 50
days. On the other hand, the pH under aerobic
conditions was slightly higher as it increased to 9
after similar incubation time and remained constant
for 50 days as well (Fig. 2D). In SRB culture
incubated under both anaerobic and aerobic
conditions, there was no detection of SRB during the
28-56 day incubation period (Fig. 3C). In contrast,
Bacterial consortia cell concentrations reached 108
CFU/mL under aerobic conditions, while SRB
reached just over 105 CFU/mL, after incubation for
28 days. As for the sulfate concentration, it remained
around 1400mg/L throughout the experiment. The
increase of SRB at 7 day incubation time indicated
that SRB was not dormant and sulfate-reducing
activity occurred during this time. This observation
suggested that sulfur-oxidizing bacteria (SOB)
generated sulfate and lived symbiotically with SRB,
in addition to the oxidation of sulfide within the
dissolved oxygen.
                                                           Figure 3. Time course on the concentration of
                                                           aerobes (A), anearobes (B), and SRB (C). Incubation
                                                           was carried out under anaerobic (  , ) and aerobic
                                                           ( , ) conditions.           , :In SRB culture,    ,
                                                           : In bacterial consortia. Cell concentrations were
                                                           meausred over a 60 day (d) period.

                                                           3.3 Characteristic of SRB colonies
                                                           For the counting of SRB in the culture of Bacterial
                                                           consortia, the culture broth was inoculated into
                                                           MBM agar plate under anaerobic condition. After
                                                           incubation for 7 days, a black colony was observed in
                                                           the plate. Following this period, the entire plate
                                                           became black due to H2S produced from the SRB
                                                           colony.    Clear zones surrounding the bacterial
                                                           colonies in the FeS on the MBM agar plates were
                                                           observed (Fig. 4).




                                                                                                 620 | P a g e
Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623




Figure 4. Clear zones surrounding the bacterial
colonies in the FeS on the MBM agar plates
incubated under anaerobic conditions.

3.4 Effects of Water Content on carbon steel coupons
      roughness
         In case of 60% WC, the boundary line
between water phase and air phase was observed at
the center of the carbon steel coupons. Yet, for 20%
WC, the boundary line was not observed on the
coupons. Examination of the eroded region on the
corroded coupon in terms of 60% WC, it was limited
to the lower half (Fig. 5C). On the other hand, in the
case of 20% WC, the eroded region was observed
over the whole area on the coupons. Shiny metal
surfaces remained after incubation for 56 days (Fig.
5D). In comparison with 100% WC, the eroded
region was observed over the entire coupon, and
developed pitting was observed (Fig. 5B). The CMD        Figure 5. Surface roughness analysis of corroded
under sterile conditions with 60% WC reached             carbon steel coupons immersed in bacterial consortia
22.5µm and was 4.4 times higher than 100% WC             under 100, 60 and 20% WC conditions. Corrosion
(5.11µm) after incubation for 56 days (Fig. 6B, Table    products were selectively removed and the roughness
2). CMD was directly proportional to the incubation      of the metal surface was analyzed. The control
time (R2= 0.993). CMD under sterile conditions with      coupon was immersed in sterile conditions. Under
20% WC reached 30.0µm and was 5.9 times more             60% WC condition, the lower half of coupon was
than 100% WC after incubation for 56 days. The           eluted. Under 20% WC condition, shiny metal
CMD was directly proportional to the incubation time     surfaces were observed.
(R2 = 0.999).




                                                         Figure 6. Time course of CMD in bacterial consortia




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Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623
(    ,   , ) and sterile conditions ( ,    , ) with      carried out under 100% WC ( ), 60% WC ( ), and
100% WC ( ,          ), 60% WC ( ,       ), and 20%      20% WC ( ). Cell concentrations were meausred
WC ( , ).                                                over a 60 day (d) period.
 Cell concentrations were meausred over a 60 day (d)
period.                                                                 IV. Discussion
                                                                   Aerobic conditions accelerated the corrosion
                                                         of carbon steel in model soil. SRB inoculation
Table 2. Effect of Water Content on Endpoint CMD         inhibited the corrosion under aerobic conditions at
        (µm) after 56 days Incubation                    day 14 and 28. The whole surfaces of the corroded
                        Bacterial              Sterile   steel coupons were covered with homogeneous
                        consortia                        sulfide (FeS). Ma et al. reported that corrosion of
                                                         99.99% pure iron immersed in solution was inhibited
  100% WC                  34.1                 5.11     by a protective layer of FeS under such conditions as
                                                         less than 0.04 mmol dm-3 H2S concentration, a pH
   60% WC                  35.9                 22.5     value of 3-5 and an immersion time longer than 2 h
                                                         [13]. When the entire coupon surface was covered
   20% WC                  37.1                 30.0     with homogeneous ferrous sulfide, corrosion of
                                                         carbon steel was inhibited. Under anaerobic or
                                                         aerobic condition, sulfate concentration for the SRB
Bacterial consortia accelerated the corrosion of the
                                                         inoculated condition stopped decreasing at day 7
carbon steel coupons under three WC conditions.          because of either the product (H2S) inhibition or
CMDs in the cases of 100, 60 and 20% WC reached
                                                         lactate depletion, respectively. The observation
34.1, 35.9 and 37.1µm, respectively after incubation
                                                         under anaerobic condition indicated that hydrogen
for 56 days (Fig. 6A, Table 2). In the case of 100%
                                                         sulfide didn`t accelerate corrosion of carbon steel.
WC, CMD was slightly increased until the 14th
                                                         Corrosion acceleration by SRB activity such as the
incubation day and the corrosion rate was accelerated    cathodic depolarization was stopped at day 7.
after that. No distinct difference in CMD among the
                                                         Bacterial consortia accelerated the corrosion of
three WC conditions was observed nor was there any
                                                         carbon steel. However, in the soil contained water,
detection of SRB after incubation for 56 days. The       little convection occurred. Therefore, segregation of
concentrations of aerobes and anaerobes decreased        bacterial habitat and uneven distribution of dissolved
under incubation with all three WC conditions (Fig.      oxygen in the culture were considered to be formed
7).
                                                         in the soil. It is indicated that the heterogeneous
                                                         structure led to the formation of a heterogeneous
                                                         biofilm with corrosion products on the carbon steel
                                                         coupons. The heterogeneous biofilm that resulted in
                                                         the uneven distribution of dissolved oxygen on the
                                                         metal surface and the formation of oxygen
                                                         concentration cells accelerated microbial corrosion.
                                                         Therefore, the heterogeneity caused acceleration in
                                                         model soil. Dubiel et al. reported that the corrosion
                                                         of carbon steel in the culture inoculated with both
                                                         SOB and SRB was accelerated compared to that in
                                                         the culture inoculated with either SOB or SRB [14].
                                                         In our experiments, SRB colonies and FeS were
                                                         observed on MBM agar plates inoculated with
                                                         Bacterial consortia culture. The entire plate became
                                                         black due to H2S produced by the SRB colony. Clear
                                                         zones surrounding the bacterial colonies in the FeS
                                                         were observed.       On the carbon steel coupons
                                                         immersed into Bacterial consortia, the protective FeS
                                                         film formed by SRB was broken by the bacteria
                                                         observed in the clear zone. The bacteria were
                                                         considered to be SOB and the mechanism of
                                                         corrosion was proposed in Fig. 8. That is being, (1)
                                                         SRB generate H2S from sulfate, (2) carbon steel
                                                         coupons are protected by FeS, (3) SOB break the
                                                         protective FeS film and sulfate is generated.
Figure 7. Time course of concentration of aerobes
(A), anaerobes (B), and SRB (C). Incubation was


                                                                                                622 | P a g e
Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications
                   (IJERA)          ISSN: 2248-9622         www.ijera.com
                        Vol. 3, Issue 2, March -April 2013, pp.617-623

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                                                                 D.K. Newman, Microbial Iron Respiration Can
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steel with protective ferrous sulfide film. Uneven
distribution of dissolved oxygen on the carbon steel,
resulting from the heterogeneity of bacterial consortia
habitat, accelerated the corrosion of carbon steel.




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Cx32617623

  • 1. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 Sulfur cycle of microbial corrosion on carbon steel in soil model Mataqi, K.Y. *, Akbar, B.H.** *(Department of Biotechnology, Kuwait Institute for Scientific Research, Kuwait) ** (Department of Biotechnology Kuwait Institute for Scientific Research, Kuwait) ABSTRACT which either oxidize or reduce sulphur compounds as This study examined the effects of some part of their life process. Although many Desulfovibrio desulfricans and bacteria consortia culturable bacterial types with known corrosion on the rate of carbon steel corrosion in soil model. effects have been identified [5], in both aquatic and Microbial corrosion was measured using the terrestrial environments the primary corrosion- corroded mean depth after 56 days incubation causing bacteria are the sulfate-reducing iron- under aerobic and anaerobic conditions. The oxidizing bacteria. Desulfovibrio desulfricans is effects of water content and dissolved oxygen in examined in this report as a source of Sulfate soil on the corrosion rate were also analyzed. reducers, which is found to exist in all soil and water Results showed that aerobic conditions increased types, as well as lives symbiotically with facultative corrosion rate. Moreover, sole Desulfovibrio anaerobic bacteria [6]. Sulfate-reducing bacteria desulfricans treatment ceased the corrosion as a (SRB) are a group of anaerobic diverse organisms in protective ferrous sulfide film formed on the which have varied morphological and nutritional carbon steel. While the heterogeneous biofilm of characteristics. They utilize organic matter to produce the bacterial consortia formed uneven oxygen sulfide by either reducing or oxidizing sulfate concentration which accelerated the corrosion. compounds [7] , as a source of energy. Therefore, sulphate (SO42–) can be reduced to sulphide (S2–) by Keywords - Bacteria , Carbon steel, Corrosion, SRB leading to the generation of hydrogen sulfide as SRB, Desulfovibrio desulfricans a metabolic bi-product. Both physical and chemical processes transfer Hydrogen sulfide (H2S) across the I. Introduction air and water boundaries to environments where Corrosion is an ever-present degradation mechanism chemoautotrophic bacteria oxidize the sulfide to in wetted components and systems. There are many sulfuric acid [8]. The corrosion process will hence forms of corrosion in metals, that include; pitting, occur by the reaction of the biogenic sulfuric acid stress corrosion, general corrosion, galvanic with the metallic surfaces [9]. In order to evaluate the corrosion, and others [1], in which microbiologist significance of microbial corrosions, a look at the have recognized and are constantly addressing. economical perspective on such effects is essential. When a system ` first encounters microbial corrosion In 2001, the cost of microbial-influenced corrosion (MC) such event usually occurs during the system on oil and gas industries accounted for about $2 initial exposure to an aqueous environment, such as billion annually [10]. Microbial induced corrosion in during hydrotest, wet lay-up, or moist soil. The the US economy has reached $350 billion annually as presence of certain bacteria in an environment will of 2010 [11]. Mild steel in used widely in piping lead to the production of microbial corrosions. This systems, storage tanks, cooling towers and aquatic mode of corrosion can be accelerated by microbial structures and is the most readily corroded metals organisms, either because they manufacture [12]. Since bacteria form colonies beneath which aggressive species, such as protons or sulphide ions, corrosion can occur, prevention of colonization is one or because they catalyze the electrochemical of the potential ways to prevent MC. This report will reactions themselves [2]. MC induction also requires examine the effects of Desulfovibrio desulfricans and the presence of nutrients and water to ensure the bacteria consortia on the rate of carbon steel survival and growth of microorganisms. Thus, the corrosion in soil model with respect to dissolved ability of microorganisms to sense and rapidly oxygen and water content distribution in such a response to harsh environmental changes is vital for system. their survival and MC capabilities [3]. Microbes can grow in fluids with pH values ranging from -1 to 10, II. Materials and Methods where -1 is the most acidic concentration, and derive 2.1 Organisms energy from organic or inorganic materials. The sulfate reducing bacterial stain Moreover, microbes can survive temperatures which Desulfovibrio desulfricans DSMZ 642 was used as range from -4 to 210oF (-20 to 99oC) [4]. The the model SRB in this study. Activated sludge from a majority of the active organisms involved in municipal wastewater plant was used as model corrosion are bacteria, about 1-5 micrometers long, Bacteria consortia. 617 | P a g e
  • 2. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 2.2 Culture medium 30mL vial bottle; (2) butyl rubber plug; (3) aluminum Modified Baar`s Medium (MBM), containing (in cap; (4) Na2S solution in 500µL microtube; (5) g/L) anhydrous sodium citrate 5.0, 50% sodium carbon steel coupons; (6) culture medium in SiO2 lactate 4.9, yeast extract 1.0, NH4Cl 1.0, soil. (B) Aerobic conditions: (7) 50mL centrifuge MgSO4.7H2O 2.0, CaSO4 1.0, and K2HPO4 0.5, was tube; (8) silicone plug. used as culture medium. The pH, sulfate To create artificial model soil, 30mL vials (Nichiden concentration and total organic carbon concentration Rika Garasu, Hyogo) containing 25g silicon dioxide (TOC) of MBM are 7.0, 1500 mg/L, and 2500 mg/L, (SiO2) sand, a 500µL microtube, and three carbon respectively. SRB and activated sludge were pre- steel coupons were autoclaved, sealed with a butyl incubated in MBM at 37oC and 25oC, respectively, rubber plug and an aluminum cap, and replaced with for seven days. Then, one-tenth of these pre- N2 gas (N2 >99.9995%) (Figure, 1A). To maintain incubated culture broths were inoculated into test the anaerobic conditions throughout the experiment, culture medium. The inoculated SRB size was 4x105 400µL of 27.2g/L Na2S solution was poured into the colony forming unit (CFU) CFU/mL D. desulfricans microtube. Then, 7.5mL of the degassed culture and 3x107 CFU/mL facultative anaerobic bacteria. medium was inoculated. To maintain aerobic The inoculated activated sludge size was 7500mg/L conditions, a 50mL centrifuge tube containing 25g mixed liquor suspended solid (MLSS). Sterile MBM SiO2 and three carbon steel coupons was autoclaved. was prepared by autoclaving the medium at 120̊ C for Followed by the inoculation with 7.5mL of the 20min. culture medium (Fig, 1B). 2.3 Carbon steel coupons preparation 2.6 Measurement of water content on corrosion Rectangular carbon steel coupons (20x10mm and To investigate the effects of water content on 0.35mm thick) were cut from a sheet stock. The corrosion, 4.5 or 1.5mL culture medium was composition of the carbon steel coupons was (in wt inoculated. Water content (WC) was defined as the %) 99.71 Fe, 0.03 C, 0.01 Si, 0.19 Mn, 0.013 P, ratio of water volume to void of the model soil. 0.017 S, 0.0017 N, and 0.026Al. The surface was Since the void of 25g SiO2 7.5mL, addition of 7.5, wet-polished with 800-grid polishing paper. The 4.5 and 1.5mL of culture medium resulted in 100, 60 polished coupons were cleaned ultrasonically in and 20% WC, respectively. To keep the water acetone for 15min, weighed, air-dried, and stored in a constant in the test tube, sterile water was added and desecrator. the tube was centrifuged (1500G, 3min) every week. 2.4 Corrosion Measurements After incubation for 0, 7, 14, 28, and 56 d, saline The corroded mean depth (CMD) was used as an (NaCl 8.0 g/L, KCl 0.2 g/L) was added to the model indicator of the extent of corrosion. Corrosion soil. The model soil was sonicated for 20 sec, and products were selectively removed from tested mixed with a spatula. coupons by incubating at 60oC for 60 min in a 10wt% 2.7 Measurement of Total Organic Carbon and HCl solution with 0.3vol% Ibit, a polycationic amine Sulfate Concentrations derivative that protects metal steel surface from HCl. Sulfate concentration and TOC concentration in the Then the treated coupons were weighed. Mass loss culture was measured by sulfur analyzer Antek (9000 of the carbon steel coupon was divided by the Series) and a TOC analyzer, respectively. The pH of specific gravity of iron (7.86g/cm3) and by the area the culture tenfold-diluted solution was measured by (cm2) of the carbon steel coupon, and CMD was a pH meter. Corrosion products were selectively derived. removed from the corroded carbon steel coupons as 2.5 Model soil preparation described above. The roughness of the coupon was analyzed by a laser 3D profile microscope (VK- 8500). 2.8 Bacterial count To count the aerobes in the culture, saline was added to the model soil and the supernatant was spread- plated on Minimized Luria-Bertani (MLB) agar plate, containing (in g/L) polypeptone 1.0, yeast extract 0.5, NaCl 10.0, and agar 15, in triplicate. After incubation for 7 d at 28oC, the observed colonies were counted as aerobes. For the counting of anaerobes and SRB in the experimental culture, the supernatant was added dropwise to the plates and the MBM agar medium with 173 (in g/L) Fe(NH4)2(SO4)2 was poured. The plates were incubated in sealed jars with an oxygen absorbing Figure 1. Experimental apparatuses for preparation of and carbon dioxide generating agent. After artificial model soil. (A) Anaerobic conditions: (1) incubation for 14 d at 37oC, the observed black 618 | P a g e
  • 3. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 colonies were counted as SRB and others were Figure 2. Time Course of CMD (A), sulfate counted as anaerobes. The concentration of bacteria concentration (B), TOC concentration (C) , and pH was expressed as (CFU) per mL. (D) under anaerobic ( , , , ) and aerobic ( , , ) conditions. The test was conducted in SRB culture III. Results ( , ), bacterial consortia ( , ) and in sterile 3.1 Analysis of corroded carbon steel conditions as a control ( , ). The CMDs of carbon steel coupons under anaerobic and aerobic conditions were compared after 56 days incubation period. In Bacterial Table 1. Effect of dissolved Oxygen Existence and consortia, the CMD under aerobic conditions reached Bacteria on Endpoint CMD (µm) after 56 days 34.1 µm, which was 28 times higher than anaerobic Incubation conditions with CMD measurement of only 1.2 µm. SRB Bacterial Sterile However, the CMD for SRB-inoculation or sterile consortia conditions reached 5.42 or 5.11 µm respectively, in which was 8.9 or 8.8 times higher than the anaerobic conditions (Fig. 2A, Table 1). There weren’t any Anaerobic 0.61 1.2 0.58 significant differences between SRB and sterile control conditions, yet in Bacterial consortia a change was noted with 2.1 and 6.7 times increase in Aerobic 5.42 34.1 5.11 anaerobic and aerobic treatments respectively, when compared with sterile CMD values. These observations indicated that existence of oxygen Upon inoculation of SRB under aerobic conditions, causes carbon steel corrosion in model soil. little corrosion on the steel coupons was observed during the 14-28 days incubation period. In the model soil analyzed, a gap separating the soil into upper and lower parts was observed just after incubation for 7 days. This gap seemed to influence corrosion acceleration, as the coupons were corroded unexpectedly at day 7. The whole surface of coupon immersed into the SRB culture was covered with homogeneous black corrosion products that were easily wiped off. When the corroded coupons were incubated with HCl, hydrogen sulfide (H2S) gas was produced, indicating that FeS was formed. In addition, local corrosion under black tubercles was observed under aerobic conditions. These observations showed that the sole existence of SRB didn`t accelerate carbon steel corrosion, while that of Bacterial consortia did indeed accelerate the corrosion process. 3.2 Influence of pH, Sulfate and TOC concentrations on bacterial growth over time Sulfate concentration for the SRB inoculated in both anaerobic and aerobic conditions stopped decreasing at day 7 suggesting that the sulfate-reducing activity of SRB was high until this day as it began to decrease afterwards. While, the sulfate concentration for Bacterial consortia stopped decreasing at day 14 (Fig. 2B). Incubation of carbon steel coupons with SRB or Bacterial consortia inoculae lead to the depletion of TOC concentration at day 7; furthermore, heterotrophic bacteria were inactive during the 14-56 day incubation period (Fig. 2C) and the concentration of aerobes decreased as well (Fig. 3A). Under anaerobic conditions, the TOC was not consumed completely, because oxygen as electron acceptor was limited. TOC concentration decreased even under sterile conditions. In another experiment with sterile conditions that did not contain carbon 619 | P a g e
  • 4. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 steel coupons, the TOC concentration however did not decrease (data not shown). This indicates that the corrosion products adsorbed the organic carbon. Under anaerobic conditions, SRB concentration was decreased and became negligible during the 28-56 day incubation period (Fig. 3C). This is due to the fact that insufficient sulfate and hydrogen sulfide that filled the tested bottle inhibited the growth of SRB. Under aerobic conditions, the presence of SRB suggested the formation of anaerobic regions in the soil. When examining the pH concentration under anaerobic conditions, it increased to 7.9 after incubation for 7 days and remained constant for 50 days. On the other hand, the pH under aerobic conditions was slightly higher as it increased to 9 after similar incubation time and remained constant for 50 days as well (Fig. 2D). In SRB culture incubated under both anaerobic and aerobic conditions, there was no detection of SRB during the 28-56 day incubation period (Fig. 3C). In contrast, Bacterial consortia cell concentrations reached 108 CFU/mL under aerobic conditions, while SRB reached just over 105 CFU/mL, after incubation for 28 days. As for the sulfate concentration, it remained around 1400mg/L throughout the experiment. The increase of SRB at 7 day incubation time indicated that SRB was not dormant and sulfate-reducing activity occurred during this time. This observation suggested that sulfur-oxidizing bacteria (SOB) generated sulfate and lived symbiotically with SRB, in addition to the oxidation of sulfide within the dissolved oxygen. Figure 3. Time course on the concentration of aerobes (A), anearobes (B), and SRB (C). Incubation was carried out under anaerobic ( , ) and aerobic ( , ) conditions. , :In SRB culture, , : In bacterial consortia. Cell concentrations were meausred over a 60 day (d) period. 3.3 Characteristic of SRB colonies For the counting of SRB in the culture of Bacterial consortia, the culture broth was inoculated into MBM agar plate under anaerobic condition. After incubation for 7 days, a black colony was observed in the plate. Following this period, the entire plate became black due to H2S produced from the SRB colony. Clear zones surrounding the bacterial colonies in the FeS on the MBM agar plates were observed (Fig. 4). 620 | P a g e
  • 5. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 Figure 4. Clear zones surrounding the bacterial colonies in the FeS on the MBM agar plates incubated under anaerobic conditions. 3.4 Effects of Water Content on carbon steel coupons roughness In case of 60% WC, the boundary line between water phase and air phase was observed at the center of the carbon steel coupons. Yet, for 20% WC, the boundary line was not observed on the coupons. Examination of the eroded region on the corroded coupon in terms of 60% WC, it was limited to the lower half (Fig. 5C). On the other hand, in the case of 20% WC, the eroded region was observed over the whole area on the coupons. Shiny metal surfaces remained after incubation for 56 days (Fig. 5D). In comparison with 100% WC, the eroded region was observed over the entire coupon, and developed pitting was observed (Fig. 5B). The CMD Figure 5. Surface roughness analysis of corroded under sterile conditions with 60% WC reached carbon steel coupons immersed in bacterial consortia 22.5µm and was 4.4 times higher than 100% WC under 100, 60 and 20% WC conditions. Corrosion (5.11µm) after incubation for 56 days (Fig. 6B, Table products were selectively removed and the roughness 2). CMD was directly proportional to the incubation of the metal surface was analyzed. The control time (R2= 0.993). CMD under sterile conditions with coupon was immersed in sterile conditions. Under 20% WC reached 30.0µm and was 5.9 times more 60% WC condition, the lower half of coupon was than 100% WC after incubation for 56 days. The eluted. Under 20% WC condition, shiny metal CMD was directly proportional to the incubation time surfaces were observed. (R2 = 0.999). Figure 6. Time course of CMD in bacterial consortia 621 | P a g e
  • 6. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 ( , , ) and sterile conditions ( , , ) with carried out under 100% WC ( ), 60% WC ( ), and 100% WC ( , ), 60% WC ( , ), and 20% 20% WC ( ). Cell concentrations were meausred WC ( , ). over a 60 day (d) period. Cell concentrations were meausred over a 60 day (d) period. IV. Discussion Aerobic conditions accelerated the corrosion of carbon steel in model soil. SRB inoculation Table 2. Effect of Water Content on Endpoint CMD inhibited the corrosion under aerobic conditions at (µm) after 56 days Incubation day 14 and 28. The whole surfaces of the corroded Bacterial Sterile steel coupons were covered with homogeneous consortia sulfide (FeS). Ma et al. reported that corrosion of 99.99% pure iron immersed in solution was inhibited 100% WC 34.1 5.11 by a protective layer of FeS under such conditions as less than 0.04 mmol dm-3 H2S concentration, a pH 60% WC 35.9 22.5 value of 3-5 and an immersion time longer than 2 h [13]. When the entire coupon surface was covered 20% WC 37.1 30.0 with homogeneous ferrous sulfide, corrosion of carbon steel was inhibited. Under anaerobic or aerobic condition, sulfate concentration for the SRB Bacterial consortia accelerated the corrosion of the inoculated condition stopped decreasing at day 7 carbon steel coupons under three WC conditions. because of either the product (H2S) inhibition or CMDs in the cases of 100, 60 and 20% WC reached lactate depletion, respectively. The observation 34.1, 35.9 and 37.1µm, respectively after incubation under anaerobic condition indicated that hydrogen for 56 days (Fig. 6A, Table 2). In the case of 100% sulfide didn`t accelerate corrosion of carbon steel. WC, CMD was slightly increased until the 14th Corrosion acceleration by SRB activity such as the incubation day and the corrosion rate was accelerated cathodic depolarization was stopped at day 7. after that. No distinct difference in CMD among the Bacterial consortia accelerated the corrosion of three WC conditions was observed nor was there any carbon steel. However, in the soil contained water, detection of SRB after incubation for 56 days. The little convection occurred. Therefore, segregation of concentrations of aerobes and anaerobes decreased bacterial habitat and uneven distribution of dissolved under incubation with all three WC conditions (Fig. oxygen in the culture were considered to be formed 7). in the soil. It is indicated that the heterogeneous structure led to the formation of a heterogeneous biofilm with corrosion products on the carbon steel coupons. The heterogeneous biofilm that resulted in the uneven distribution of dissolved oxygen on the metal surface and the formation of oxygen concentration cells accelerated microbial corrosion. Therefore, the heterogeneity caused acceleration in model soil. Dubiel et al. reported that the corrosion of carbon steel in the culture inoculated with both SOB and SRB was accelerated compared to that in the culture inoculated with either SOB or SRB [14]. In our experiments, SRB colonies and FeS were observed on MBM agar plates inoculated with Bacterial consortia culture. The entire plate became black due to H2S produced by the SRB colony. Clear zones surrounding the bacterial colonies in the FeS were observed. On the carbon steel coupons immersed into Bacterial consortia, the protective FeS film formed by SRB was broken by the bacteria observed in the clear zone. The bacteria were considered to be SOB and the mechanism of corrosion was proposed in Fig. 8. That is being, (1) SRB generate H2S from sulfate, (2) carbon steel coupons are protected by FeS, (3) SOB break the protective FeS film and sulfate is generated. Figure 7. Time course of concentration of aerobes (A), anaerobes (B), and SRB (C). Incubation was 622 | P a g e
  • 7. Mataqi, K.Y. , Akbar, B.H. / International Journal of Engineering Research and Applications (IJERA) ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 2, March -April 2013, pp.617-623 References [1] S. Virtanen, I. Milosev, E. Gomez-Barrena, R. Trebse, J. Salo, and YT. Konttinen, Special modes of corrosion under physiological and simulated physiological conditions. Acta Biomater 4(3), 2008, 468-76. [2] R. B. Eckert, H. C. Aldrich, C. A. Edwards, B. A. Cookingham, Microscopic Differentiation of Internal Corrosion Initiation Mechanisms in Natural Gas Pipeline Systems. Corrosion, 2003, 1342-1355. Figure 8. Sulfur cycle with SRB and SOB [3] R. Javaherdashti , Impact of sulphate-reducing accelerating carbon steel corrosion. bacteria on the performance of engineering materials. Appl Microbiol Biotechnology 91(6), 2011, 1507-17. The corrosion rate of carbon steel coupons [4] A. G. Brooke, E.M. Watling, M. Attwood, increased in the order of 20, 60, and 100% WC. In M.&Tempest, D. W. Environmental Control of the case of 60% WC, boundary line between the Metabolic Fluxes in Thermotolerant Methylotrophic water phase and the air phase was observed at the Bacillus Strains. Archives of Microbiology. 151, 1989, 268-273. center of carbon steel coupons. Above the water [5] RA . Lane, Under the microscope: understanding, phase, the soil had adhering water. In the case of detecting and preventing microbiologically 20% WC, corrosion that begun from adhering water influenced corrosion. AMPTIAC Quart 9(1), 2005, extended to cover the entire carbon steel coupons. In 3–8. the case of 60% WC, the corrosion was observed not [6] K. R. Butlin, M. E. Adams, and M. Thomas, The on the upper half of coupon but on the lower half. isolation and cultivation of sulfate-reducing bacteria. The difference in dissolved oxygen concentration J. Gen. Microbiology 3, 1949, 46-59. between the parts under adhering water and the metal [7] G.R. Gibson, Physiology and ecology of the surface on the upper half of the coupon was less than sulphatereducing bacteria. J Appl Bacteriology 69, 1990, 769–797. that between the lower half and the upper half of the [8] M.S. Wiener, B.V. Salas, M. Quintero-Nunez, and coupons. Therefore, the oxygen concentration cell R. Zlatev. Effect of H2S on corrosion in polluted reaction was limited between the lower half and the waters: a review. Corros. Eng. Sci. Technol. 41 (3), upper half of the coupon. The uneven distribution of 2006, 221–227. water and air spaces on the carbon steel coupons [9] M. Magot, G. Ravot, X. Campaignolle, B. Ollivier, accelerated the corrosion. BKC. Patel , ML. Fardeau, P.Thomas, JL. Crolet, and In Bacteria consortia, the corrosion of carbon steel JL. Garcia, Dethiosulfovibrio peptidovorans gen. coupons under 60 and 100% WC conditions was nov., sp. nov., a new anaerobic, slightly halophilic, thiosulfate-reducing bacterium from corroding accelerated to almost same level as the corrosion offshore oil wells. Int J Syst Bacteriol 47, 1997, 818– observed under the 20% WC condition. Therefore, 824. the heterogeneity resulting from bacteria led to an [10] G.H. Koch, M. P. H. Brongers, N. G. Thompson, Y. uneven distribution of dissolved oxygen on the P. Virmani, and J. H. Payer, Corrosion costs and carbon steel coupon, which is equal to the preventive strategies in the United States. 2001. distribution under the 20% WC condition, and that FHWA-RD-01–156. [Online.] Federal Highway was the major mechanism of microbial corrosion Administration, Washington,D.C. acceleration. http://www.corrosioncost.com/. [11] E. Rob, Biological corrosion of metals. Faliure Analysis. 2010. [Online.] http://failure- V. Conclusion analysis.info/2010/06/biological-corrosion-of- Carbon steel coupons were immersed into metals/. artificial model soil consisting of silica sand, [12] E.J. Akpabio, E.J . Ekott, and M.E. Akpan, Inhibition microbes, and medium. Incubation was carried out and control of microbiologically influenced corrosion under anaerobic and aerobic conditions, and aerobic in oilfield materials. Environmental Research conditions were found to accelerate corrosion of Journal 5(2), 2011, 59-65. carbon steel. Carbon steel coupons were immersed [13] H. Ma, X. Cheng, S. Li, Z. Chen, S. Quan, L. Zhao, into artificial model soils with 100, 60 and 20% WC. and S. Niu, Corrosion Science. 42, 2000, 1669-1683. The existence of air space and the uneven distribution [14] M. Dubiel, C. H. Hsu, C. C. Chien, F. Mansfeld, and D.K. Newman, Microbial Iron Respiration Can of medium on the carbon steel coupons accelerated Protect Steel from Corrosion. Applied and corrosion. Sole SRB ceased the corrosion of carbon Environmental Microbiology 68, 2002,1440-1445. steel with protective ferrous sulfide film. Uneven distribution of dissolved oxygen on the carbon steel, resulting from the heterogeneity of bacterial consortia habitat, accelerated the corrosion of carbon steel. 623 | P a g e