Disinfection of Sulfate Reducing Bacteria using Ultraviolet Treatment in Mitigating Microbiologically Influenced Corrosion
Akrima Abu Bakar,
Rosilawati Mohd Rasol,
Muhammad Khairool Fahmy,
Siti Nor Fariza
Ahmad Safuan A. Rashid
Oil and gas pipeline systems often fail due to Microbiologically Influenced
Corrosion (MIC). Chemical biocide is one of the common treatments to combat
MIC. However, this method is costly and further usage of chemical biocides may
lead to serious environment pollutions. In an attempt to disinfect Desulfovibrio
vulgaris, a species involved in MIC from oil and gas pipelines, ultraviolet
wave is used as a potential alternative. D. vulgaris (ATCC7757) was cultured
in broth number 1249 (Modified Barrs Medium) and the medium was adjusted
to pH 9.5 at 37°C based on the Sulfate Reducing Bacteria (SRB) optimum growth
parameters. Carbon steel coupons grade API 5L X70 were cut to approximately
10x20x5 mm and kept in anaerobic vials along with the cultured medium. The prepared
samples were incubated for 11 days, followed by exposure to ultraviolet for
1 h. Results from the study elucidated that ultraviolet (UV) is a viable option
for SRB disinfection and could effectively reduce the growth of SRB.
to cite this article:
Akrima Abu Bakar, Norhazilan Noor, Nordin Yahaya, Rosilawati Mohd Rasol, Muhammad Khairool Fahmy, Siti Nor Fariza and Ahmad Safuan A. Rashid, 2014. Disinfection of Sulfate Reducing Bacteria using Ultraviolet Treatment in Mitigating Microbiologically Influenced Corrosion. Journal of Biological Sciences, 14: 349-354.
Received: November 05, 2013;
Accepted: February 21, 2014;
Published: April 15, 2014
Corrosion is one of the natural deterioration phenomenon that will affect financial
and safety implications in oil and gas industry. Pipeline systems that are used
to convey crude oils over long distances have always been facing serious corrosion
problems that can lead to system failures (Noor et al.,
2007). The failure investigation results indicated that the pipes have most
likely suffered from severe corrosion damage due to Microbiologically Influenced
Corrosion (MIC) (Al-Sulaiman et al., 2010). Microbial
activity in any environment occurs in the presence of water, a carbon source,
an electron donor and an electron acceptor, all of which can be present in oil
pipelines (Almahamedh et al., 2011).
MIC is well known as one of the major causes that will accelerate the process
of corrosion in pipelines. It implies that the increase in corrosion rate is
due to the presence of bacterial activities that accelerates the rate of anodic
and/or cathodic reactions (Mansfeld, 2007). One of
the bacterial types that can be mostly found living in the absence of oxygen
in the oil and gas pipelines is Sulfate Reducing Bacteria (SRB) or to be specific
the species known as Desulfovibrio vulgaris. SRB are obligate anaerobes
that obtain energy for growth from the oxidation of organic substances, using
sulfate as the external electron acceptor (Widdle, 1988;
Postgate, 1984). In oilfield systems, SRB can cause corrosion
of iron in the absence of air and contamination of fuel gas and stored fuel
gas with hydrogen sulfide (H2S) (Cord-Ruwisch
et al., 1987). Crudes containing H2S are termed as sour
crude and this can promote the growth of SRB. SRBs are anaerobic and do not
need oxygen to survive and they use sulfate ions as a terminal acceptor and
produce H2S (Alabbas et al., 2012).
Biocides injection is the typical technique to mitigate MIC which is toxic
and must be carefully handled cost ineffective and could cause environment pollutions.
In some countries, the use of biocides has to follow stringent regulations.
In order to minimise the use of biocides, ultraviolet technique has been introduced
to mitigate MIC. Ultraviolet is an established technology for disinfection and
controlling the bacterial populations. But the previous studies still lack optimization
of the ultraviolet dosage according to specific bacterial properties and parameters.
According to Barnard and Morgan (1903), the UV-radiation
kills the microbial cells primarily due to its action on deoxyribonucleic acid
(DNA). Bacteria, phages and viruses are also killed or reduced by photochemical
wet combustion down to or below detection limits of organic carbon.
The study presented explores the potential of ultraviolet as an inhibitor to SRB growth. D. vulgaris was cultured in a Modified Barrs Medium and samples were treated for 1 h and left incubated for 11 days. During the treatment process, samples were retrieved on several days to measure treatment efficiency. Field Emission Scanning Electron Microscopy (FESEM) was performed on the retrieved coupon before and after being exposed to SRB, to observe the effect of SRB on the carbon steel surface microstructure.
MATERIALS AND METHODS
Microorganism and media: The microorganism used in this study was SRB strain, Desulfovibrio vulgaris ATCC 7757 and cultured in a defined modified ATCC 1249 medium. Table 1 listed the composition of the modified ATCC 1249 medium. Ferrous ammonium sulfate (Fe(NH4)2(SO4)2) at 25 ppm concentration was filtered and was add-in into the sterile medium.
Materials: The material used in this study is a carbon steel coupon grade API 5L X-70. Samples were cut into 10x20x5 mm dimension to fit the anaerobic vials openings. The test coupons were polished with 100 grit Si-C paper, cleaned and dried with ethanol to remove all form of dirt, grease and small Si-C particles on the coupon surface. The cleaned and dried coupons were then coated with prime coat leaving only the top surface exposed. The coupons were then dried overnight in an oven at 37°C. The exposed area of the coupon was polished again with series of SI-C paper grade (320, 600 and 800), followed by ethanol degreasing.
Method of inoculation: All of the medium compositions were mixed with 1 L of distilled water. The pH was adjusted by using buffer chemical to 7.0 and the test medium was sterilized in an autoclaved at 121°C for about 20 min. After cooling, the medium was purged with oxygen-free nitrogen for 1 h in a laminar flow hood to remove the oxygen in the solution. The test coupons and 100 mL of medium were then transferred into the 125 mL of anaerobic and sealed with butyl rubber to maintain the anaerobic condition. Two milliliter of 1 day old SRB seed was inoculated into the medium and incubated at 37°C. The black colour of the medium is the indicator of successful inoculation within 2-3 days. FESEM on the coupons before and after being exposed to SRB was performed to observe the effect of SRB to the carbon steel surface microstructure.
|| Composition of the modified ATCC 1249 medium
Weight loss test: During the test, coupons were retrieved from anaerobic
vials every 7 days (7, 14, 21 and 28). The coupons were cleaned with cleaning
mineral solution (mixture of HCL and mineral solution) to remove all forms of
dirt. The coupons were dried and their weights before and after immersion in
the medium was recorded. Metal loss was determined by using the equation as
follows (Yahaya et al., 2011; Norhazilan
et al., 2012):
Metal loss (W) = (Wt-Wa)/A
where, Wt is initial weight of coupon (g), Wa is final weight of coupon (g) and A is area (cm2).
Ultraviolet treatment: The above mentioned sample preparation was repeated with pH and temperature set at 9.5 and 37°C, respectively. The filled anaerobic vials were exposed to ultraviolet radiation at wavelength 254 nm (contact time is 60 min) and the sample was left to incubate at temperature 37°C for 22 days. Weight of steel coupons were recorded before and after the experiment. The turbidity of the SRB was taken on a daily basis until day 11 using spectrophotometer DR5000. Serial dilution method was used to ease the measurement of turbidity of SRB. In this experiment, turbidity has been chosen as the SRB growth indicator method.
RESULTS AND DISCUSSION
The coupons for this experiment were divided into 2 categories; the samples
that had been exposed to SRB (named as SRB samples) and samples that were not
exposed to SRB (named as control samples). After immersion periods, coupons
were removed and cleaned with mineral solution and dried before weighing. Final
weights of the steel coupons were taken to calculate the weight losses.
|| Graph of weight loss (%) vs. days
||Turbidity on selected days and the difference of values between
samples untreated and treated using UV
The metal loss percent which defined by the percentage of weight loss of the
steel were 0.37, 0.54, 0.61 and 0.66% for SRB samples and 0.21, 0.35, 0.45 and
0.48% for control samples at 7, 14, 21 and 28 days, respectively.
Figure 1 displays the weight loss over time. The pH and temperature
were fixed at 7 and 37°C, respectively. The pattern of the graph shows that
metal loss volume increases over time for both samples. The rate of metal loss
(slope gradient) seems to accelerate in the first 7 days and begins to decline
thereafter due to depletion of resources for the bacteria to grow for SRB sample
coupons. Metal loss also took place for the control samples but the rate was
quite low as compared to the coupons that were immersed in SRB seed. The percentage
differences of the two categories were 0.16, 0.19, 0.16 and 0.18% on day 7,
14, 21 and 28, respectively. After day 21, the metal loss of both samples had
already started to increase slightly because of their source to induce corrosion
has diminished. Theoretically, when bacteria is attached onto a steel surfaces,
they start to form thin films known as biofilm (Kobrin, 1993)
that consist of bacterial immobilization substratum, frequently implanted in
an organic polymer matrix of microbial origin (Dexter and
LaFontaine, 1998). Biofilm that started to form can influence corrosion
in three general categories which are production of differential aeration or
chemical concentration cells, production of organic and inorganic acids as metabolic
byproducts and also production of sulfides under oxygen-free condition (Javaherdashti,
||Turbidity between samples treated and untreated using UV
When biofilm is developed, they start to change the electrochemical properties
as well as physical properties of the metal (Javaherdashti,
2011). As for the SRB, the presence of ions such as sulfates and chlorides
which were found in the medium supports the formation of biotically-generated
sulfides in the corrosion products (Alabbas et al.,
2012).Therefore, the longer the carbon steel is exposed to SRB, the more
biofilm will be produced onto the coupons surface. This will cause an increasing
amount in metal loss and cause corrosion such as pitting dependent on time.
In order to test the disinfection efficiency of UV treatment upon SRB growth,
turbidity of the SRB samples were recorded on selected days. Higher turbidity
shows the higher population of SRB presence. The characteristic of unpleasant
smells from hydrogen sulfide and black coloured solution were the evidence of
SRB growth and its metabolism in the medium (Postgate, 1984).
Table 2 lists the data of turbidity on selected days and the
difference of value between untreated and treated samples using UV. Based on
the data, it clearly shows that turbidity value of all the samples decreased
after the UV treatment.
Figure 2 show the clear view of the UV wave treatment effectiveness.
The disinfection of SRB growth using UV waves proved to be effective due to
the decrease in turbidity value after the treatment. Figure 3
illustrates the treatment efficiency in percentage over time. According to Wang
et al. (2005), UV wavelength that is considered as the most effective
germicidal with the germicidal activity peaks at 254 nm which compare well with
the UV wavelength used in this study. From the graph pattern, it indicates that
the treatment efficiency increases over time. It can be stated that the longer
the samples exposed to the UV wave, the higher the number of SRBs that could
be exterminated. The most effective treatment time is day 11 because it has
the highest difference of turbidity value.
The statistical analysis of the data is shown in Table 3 and 4. The numerical correlation value for paired sample correlation between the two paired samples for untreated and treated of metal loss and also untreated and treated of turbidity using ultraviolet is 0.989 and 0.992, respectively. From this value, indicates the presence of strong relationship between the pairs. Table 4 stated the paired samples test of T-Test. The Sig. (2-Tailed) value in the table is 0.017, Pair 1 and 0.027, Pair 2. This value is less than 0.05 so there is a statistically significant difference between the mean of metal loss and turbidity before and after the samples treated with ultraviolet.
|| Paired samples correlations
Figure 4 illustrated the Field Emission Scanning Electron
Microscopy (FESEM) image of corrosion product on the surface of coupon before
and after being exposed to SRB.
|| UV treatment efficiency vs. time
FESEM image of corrosion product on the
surface of coupon, (a) Before exposure to SRB and (b) After exposure to
SRB (corrosion product)
|| Paired samples test
Before exposure to SRB, the surface of the carbon steel had been cleaned to
a mirror surface by ethanol degreasing and the above image in Fig.
4 showed the FESEM image of carbon steel surface microstructure without
any substances. However, after 28 days of immersion in the medium that contains
SRB, the biofilm and corrosion product has been graphically traced on the coupon
surfaces. The substrate of the steel was scarcely visible since it was covered
with a porous black layer. The black layer was the evidence of SRB metabolism
activities on the metal surface. The jelly-glue substance, a by-product of corrosion
was also traced by FESEM. In order for MIC to happen, the corrosion process
starts with the biofilm formation on the metal surface. SRB cells will attach
to the coupon surface, grow, reproduce and produce an Extracellular Polymer
Substance (EPS). According to previous research reports, normally the EPS and
corrosion products occupied 75-95% of biofilm volume while 5-25% is occupied
by the cells (Alabbas et al., 2012).
Based on these results, it is reasonable to assume that SRB can cause severe corrosion damage on the Carbon Steel Grade API 5L-X70. The metal loss experiment has indicated that metal loss increases linearly over time of exposure to the SRB. Turbidity measurement has proved that UV wave could mitigate the growth of SRB. The implementation of UV wave during the treatment process can be one of the potential Non-physical Inhibitory (NPI) method that is cordial to the environmental, cost effective and helps in reducing chemical substances usage to disinfect microorganism specifically SRB.
The study was financially supported by Universiti Teknologi Malaysia (Grant No. GUP 03H49), the Ministry of Science and Technology of Malaysia, MOSTI (Grant No. ScienceFund 4S019) and the Ministry of Education of Malaysia, MOE (Grant No. ERGS 4L090).
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