Neem extract as an inhibitor for biocorrosion influencedby sulfate reducing bacteria: A preliminary investigation

Shaily M. Bhola a,⇑, Faisal M. Alabbas a, Rahul Bhola a, John R. Spear b, Brajendra Mishra a,David L. Olson a, Anthony E. Kakpovbia c

a Department of Metallurgical and Materials Engineering, Colorado School of Mines, Golden, CO 80401, USAb Department of Civil and Environmental Engineering, Colorado School of Mines, Golden, CO 80401, USAc Inspection Department, Saudi Aramco, Dhahran 31311, Saudi Arabia

a r t i c l e i n f o

Article history:Received 19 June 2013Received in revised form 17 September 2013Accepted 20 September 2013Available online 2 October 2013

Keywords:Neem extractBiocorrosionSRBLinepipe steel

a b s t r a c t

This work investigates the inhibition effect of Neem (Azadirachta indica) extract onmicrobiologically influenced corrosion (MIC) of API 5L X80 linepipe steel by a sulfate-reducing bacterial (SRB) consortium. The SRB consortium used in this study included threephylotypes; Desulfovibrio africanus, Desulfovibrio alaskensis and Desulfomicrobium sp. Steelcoupons were incubated in the presence of the SRB consortium without and with 4 wt.%Neem extracts for different periods of time. The morphology, compositions of the interfacesand subsequent corrosive pitting were characterized with field emission scanning electronmicroscopy (FE-SEM) coupled with energy dispersive spectroscopy (EDS). In addition, elec-trochemical impedance spectroscopy (EIS), linear polarization resistance (LPR) and opencircuit potential (OCP) were used to investigate the in situ corrosion behavior under thetwo different conditions. The results revealed that Neem extract has the capability toreduce the biocorrosion rate by approximately 50%. Neem has significantly reduced thepropensity of linepipe steel to SRB caused MIC by minimizing the cell growth and has sub-sequently suppressed the sulfide productions, sessile cell density and biofilm development.

! 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Microbiologically influenced corrosion (MIC) or biocorrosion is a considerable problem for the oil and gas industry. MIC isconsidered one of the most damaging mechanisms to pipeline steel materials. Microorganisms are thought to be responsiblefor greater than 20% of pipeline systems failures [1]. The main types of bacteria associated with metals in pipeline systemsare sulfate-reducing bacteria (SRB), iron and CO2 reducing bacteria and iron and manganese oxidizing bacteria [1,2]. Amongthese, SRB have received much attention in the oil and gas industry and MIC investigations have revealed that these micro-organisms have several detrimental metabolic activities including the ability to: (1) oxidize hydrogen as an electron donorfor metabolic life [1,2], (2) use O2 and Fe3+ as a terminal electron acceptor [3], (3) utilize aliphatic and aromatic hydrocarbonsas a carbon source [4], (4) use very low levels of water for cellular maintenance and growth [4], (5) couple sulfate reductionto the intracellular production of magnetite [4] (6) compete with nitrate-reducing/sulfur-oxidizing bacteria (NRB–SOB)(since they may have a nitrite reducing activity) [5,6] (7) and cause elemental oxidation of iron [7].

Basically, prevention and treatment of MIC is aimed mainly on destroying the microbial cell and/or preventing thedevelopment of biofilms [8]. Various commercial mitigation techniques have been used in the oil and gas industry to control

1350-6307/$ - see front matter ! 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.engfailanal.2013.09.015

⇑ Corresponding author. Tel.: +1 (303) 875 1642; fax: +1 (303) 273 3795.E-mail address: malhotra.shaily@gmail.com (S.M. Bhola).

Engineering Failure Analysis 36 (2014) 92–103

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MIC. These techniques include mechanical (i.e. pigging), chemical (i.e. biocides), electrochemical (i.e. cathodic protection)and biological (i.e. microbial injection of more beneficial microbiota) approaches [1,2,8]. Among these techniques, thebiocide is considered the most effective method. Biocides, however, are not only expensive but also pose considerable hazardto the environment and field personnel owing to their toxicity [1,2,8].

The conventional criteria governing the selection of an effective biocide include: (i) proven efficacy against a broadspectrum of microorganisms; (ii) ability to penetrate and disperse microbial slime; (iii) chemical and physical compatibilitywith other products (e.g. corrosion inhibitors) and the environment (e.g. pH effects); (iv) safe easy use and storage; (v)appropriate biodegradability; (vi) cost effectiveness [1,9,10]. Biocides, however, are inherently toxic and most of the timesdifficult to degrade. They may thus have a negative impact on the environment if used without a proper environmental riskassessment [10]. Moreover, in the past few years, the ineffectiveness of biocides against sessile organisms have beendocumented [11]. This is probably due to the inability of the chemical to penetrate thick biofilms, in addition to physiologicaldifferences between sessile and planktonic cells [12]. It has also been reported that biocides’ sensitivity can be altered up to1000-fold by changes in nutrients and growth rates [11].

Use of naturally occurring compounds such as plant extracts might be considered as an environmentally benign way oftreating MIC. A number of plant oils and aqueous plant extracts have been shown to have inhibitory activity against yeast,filamentous fungi and bacteria [13–15]. Indian species such as clove, cinnamon, horse raddish, cumin, tamarind, garlic,onion, are use as preservatives, disinfectants and antiseptics [16]. Vitamins [17] and other biomolecules [18] have also beenused as potential corrosion inhibitors for steel and nickel in acidic media.

Neem tree (Azadirachta indica) is well known for its unusual biological properties. Its bark and leaves are known to pos-sess diverse and multifarious therapeutic uses for the treatment of many diseases [19]. The most important bioactive prin-cipal constituent in Neem is Azadirachtin [16,19]. Neem leaf extract has been widely explored as a potential corrosioninhibitor for alloys such as mild steel, carbon steel and zinc in acidic medium [19–21].

Despite this knowledge, there is lack of information on the effects of Neem extract on biocorrosion. The presentinvestigation focuses on the use of Neem extract as a MIC inhibitor. The effect of 6% w/w azadirachtin towards SRB causedmicrobiologically influenced corrosion of API 5L grade X80 carbon steel has been explored.

2. Materials and methods

2.1. Organisms and culture

The SRB mixed cultures of Desulfovibrio africanus sp. (ATCC 19997), Desulfovibrio alaskensis (ATCC 14653) and Desulfomicr-obium sp. (Accession # KC756851) [7], were used in this study. Both D. africanus sp., D alaskensis were obtained in freezedried samples obtained from American Type Culture Collection (ATCC) while Desulfomicrobium sp. were isolated from watersamples obtained from an oil well located in Louisiana, USA. The SRB cultures were cultivated in supplemented enrichedartificial sea water. The growth medium was composed of magnesium sulfate (2.0 g), sodium citrate (5.0 g), calcium sulfatedi-hydrate (1.0 g), ammonium chloride (1.0 g), sodium chloride (25.0 g), di-potassium hydrogen orthophosphate (0.5 g),sodium lactate 60% syrup (3.5 g), and yeast extract (1.0 g). All components were per liter of distilled artificial seawater.The pH of the medium was adjusted to 7.5 using 5 M sodium hydroxide and sterilized in an autoclave at 121 "C for20 min. The SRB species were cultured in the growth medium with filter-sterilized 5% w/w ferrous ammonium sulfate addedto the medium at a ratio of 0.1–5.0 ml respectively. The bacteria were incubated for 72 h at 37 "C under an oxygen-free nitro-gen headspace.

2.2. Sample preparation

Pipeline steel (API 5L X80) coupons, provided by SAUDI ARAMCO, Saudi Arabia, were used for this study and composed ofthe following elements with a weight ratio of 0.073% C, 1.36% Mn, 0.004% P, 0.008% Ti, 0.003% S and balance as Fe. The steelhas already been characterized in our previous research [7].

For corrosion evaluation, the coupons were machined to a size of 10 ! 10 ! 5 mm and embedded in a mold of non-conducting epoxy resin, leaving an exposed surface area with a polished mirror finish of 100 mm2. For electrical connection,a copper wire was soldered at the rear of the coupons prior to epoxy embedding. The coupons were polished with a progres-sively finer polishing paper until a final grit size of 600 micro was obtained. After polishing, the coupons were rinsed withdistilled water, ultrasonically degreased in acetone and sterilized by exposing to pure ethanol for 24 h.

2.3. Electrochemical tests

The electrochemical measurements were made in a conventional three electrode ASTM glass cell coupled with a poten-tiostat and a high frequency impedance analyzer (Gamry-600). The electrochemical cell composed of a test coupon as aworking electrode (WE), a graphite electrode as an auxiliary electrode and a saturated calomel electrode (SCE) as a referenceelectrode. All glasswares were autoclaved at 121 "C for 20 min at 20 psi pressure and then air dried for an initial aseptic con-dition. Graphite electrodes, purging tubes, rubber stoppers and needles were sterilized by immersing in 70 vol.% ethanol for24 h followed by exposure to a UV lamp for 20 min. Two solutions were used in this experiment, without Neem (M1) and

S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103 93

with Neem (M2). Under a sterile hood (in a bio-safety cabinet), the electrochemical cells were assembled by pouring 700 mlof enriched artificial seawater as described above and inoculated with 5 ml of SRB consortium at 108 cells/ml. Neem extractobtained from Arborjet Inc. (AZASOL, comprising of 6% w/w azadirachtin) was added at 4% w/w to one of the cells. Theelectrochemical cells were purged for one hour with pure nitrogen gas to establish the anaerobic environment.

Open circuit potential values (OCP) of the systems were monitored with time during the immersion period followed byperiodic readings for up to 336 h. Impedance measurements were performed on the system at the open circuit potential forvarious time intervals from immersion up to 288 h. The frequency sweep was applied from 105 to 10"2 Hz and AC amplitudeof 10 mV. During the LPR technique, polarization resistance (Rp) was measured on the system at a scanning amplitude of±10 mV with reference to the open circuit potential for various time intervals from immersion up to 336 h.

During the tests, the bacterial activities were estimated by counting of the living planktonic cells using Petroff-Haussercounting chamber under the microscope at a magnification of 40!.

2.4. Sulfide measurements

The sulfide level in the vials was monitored over the test period to monitor the activities of SRB consortium. Samples ofthe test medium were extracted from the electrochemical cells using a sterile syringe in aseptic conditions under a sterilebio-safety cabinet/hood. The standard protocol detailed under the American Public Health Association standard wasfollowed (APHA 1989) [22].

2.5. Surface analysis and corrosion product compositions of the coupons exposed to SRB

At the conclusion of each test, the working electrode was carefully removed from the system for fixation. To fix the grownbiofilm to the steel surface, the coupons were immersed for 1 h in a 2% glutaraldehyde solution, dehydrated with 4 ethanolsolutions (15 min each) of volume%, 25%, 50%, 75% and 100% successively, air dried overnight and then gold sputtered [23].After fixation, the coupons were examined using field emission scanning electron microscopy (FE-SEM) coupled with energydispersive spectroscopy (EDS) to evaluate the morphology and chemical composition of the biofilm. The coupons were thencleaned using a standard protocol described under the ASTM standard (ASTM-GI-03) [24] and the pit morphology anddensity on the exposed coupons were examined using FE-SEM.

3. Results and discussion

3.1. Bacteria activities and sulfide measurements

Fig. 1 illustrates the growth process of SRB in the enriched artificial seawater medium prepared without Neem (M1) andwith Neem (M2). The results indicated that SRB in M1 system followed a typical bacterial growth cycle comprising of fourphases – lag phase, log/exponential phase, stationary phase and decline/death phase. During the lag phase, which was up to48 h, SRB start to colonize and gather nutrition to flourish. From 48 to 96 h, during the exponential phase, the number ofviable SRB species increased quickly to approximately the maximum value of 108 cells ml"1. It has been shown that duringthe exponential phase, the concentration of hydrogen sulfide also increases [25]. The dissolved sulfide measurements vari-ations over the period of exposure time are shown in Table 1. As shown in Table 1, the level of dissolved sulfide increaseddrastically for the first 96 h to a maximum value of 1768 lg l"1and then decreased to an approximate value of 1400 lg l"1,

20 40 60 80 100 120 140 160 180 200

2

3

4

5

6

7

8

SRB with Neem SRB Lo

g N SR

B/m

l)

Times (hours)

Fig. 1. SRB growth trend under biotic conditions with and without Neem extract.

94 S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103

which then remained more or less stable. The high production of sulfide occurred during the cellular exponential growthphase until it reached its maximum value (1768 lg l"1). After 96 h, the growth cycle reached the 3rd stage called thestationary phase which lasted from 96 to 168 h. During this phase, there was no increase in the number of cells and thegrowth was limited by insufficient nutrients and accumulations of the by-products of cellular metabolism [25,26]. Possibly,the accumulation of high concentration of sulfide, produced during the exponential phase, and the nutrient limitations mighthave hindered the growth of SRB. It was suggested that the high sulfide concentration can completely inhibit the SRB culturegrowth [27]. The inhibition may be the result of an intrinsic toxicity of H2S to living systems or it might be due to indirecttoxicity generated by rendering the iron insoluble as iron sulfide. Iron is needed as an essential cofactor to various cyto-chromes involved in cellular respiration (e.g. Cytochrome-C) [27]. After 168 h, the SRB cells declined to #107 cells ml"1,which was an indicative of death/decline phase.

The M1 solution became completely black within 72 h and remained black for the entire time of the experiment. Thecharacteristic unpleasant smell of hydrogen sulfide and the black colored solution were evident of SRB growth andmetabolism.

In contrast, in the M2 system where Neem was added, the cell count of viable SRB was about 102 cells ml"1 for almostentire duration of the experiment (Fig. 1) and the concentrations of sulfide measured were below 2 lg l"1. Probably, theNeem extract did not allow SRB to flourish through various phases of their life cycle and limited their metabolic processesto directly propagate them into the decline phase. The M2 solution had an orange color and did not turn black for the entirelength of the experiment. These observations suggest the inhibition effects of Neem extract on the growth of the SRBconsortium.

3.2. Surface morphology, elemental analysis and biofilm structures

At the conclusion of the tests, the visual inspection of the steel coupon exposed to the biotic system with no Neem (M1)revealed dense, thick and black products covering the surface while orange heterogeneous layer was covering the steelsurface exposed to the biotic system with Neem (M2). As shown in Figs. 2 and 3, there is a significant difference in theappearance, structures and morphology of the corrosion products that developed on the steel coupons exposed to the M1conditions compared to those exposed to M2.

Both the morphological FE-SEM image observations and energy dispersive spectroscopy (EDS) elemental analysis ofcorrosion products of API X80 steel immersed in the biotic system without Neem (M1) are shown in Fig. 2A, B and C. InFig. 2A, there are two distinctive regions: light region (R1) and dark region (R2). Quantitative EDS analysis shows that R1

Table 1Sulfide measurements in (lg/l) under biotic conditions with and without Neem extract.

Time (h) Biotic system without Neem (M1) Biotic system with Neem (M2)

48 425 ± 4 <272 432 ± 6 <296 1768 ± 11 <2

120 1538 ± 12 <2144 1366 ± 6 <2192 1348 ± 2 <2

(B) EDS analysis for light region (R1) (C) EDS analysis for the dark region (R2)

Light Region (R1) Dark Region (R2)

A A

B C

Fig. 2. (A) FE-SEM images for API 5L X52 surface exposed to biotic system with no Neem extract (M1), and EDS analysis for the light and dark regionrespectively.

S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103 95

have corrosion products and deposits that are composed of high amount of sulfides, oxygen, iron, and carbon in addition tophosphorous and sodium chloride salts (Fig. 2B and Table 2). The light region (R1) is considered the outer layer where thesulfide and carbon are the predominant constituents. The dark region (R2) is considered the inner layer in which the iron,oxygen and carbon are the predominant elements as shown in Fig. 2C and Table 2. The outer and inner layers were chosenbased on our experience in our previous research papers and no cross section view was performed on the sample [7]. Theelemental analysis shown in Table 2, Fig. 2A, B and C, suggest that the surface deposits and corrosion products under M1conditions might be composed of significant accumulations of iron oxides, sulfur-based, organic and phosphorous-basedcompounds.

In contrast, the FE-SEM images and EDS spectra display the morphological and chemical characteristics of the layer grownon carbon steel exposed to biotic conditions with Neem (M2) as shown in Fig. 3A, B and C. As shown in Fig. 3A, there are twodistinctive areas: white area/light region (A1) and black area/dark region (A2). The black area has homogenous, cohesivestructures while the white area has incoherent, cracked and flaky structures. Quantitative EDS analysis shows that the areaA2 is composed of a higher amount of iron, carbon, oxygen in addition to sodium chloride salts, phosphates and sulfide(Fig. 3C and Table 2). The EDS analysis suggests that the A2 is the inner region due to the iron content and presence ofmanganese, which were related to the steel constituents. The region A2 might be composed of iron oxides in addition tounknown organic compounds. The white region (A1) is considered the outer layer and composed mainly of carbon, iron,oxygen, and sodium chloride salts, Fig. 3B. The EDS elemental analysis shown in Fig. 3B and Table 2, suggest that this layermight compose mainly of iron oxides mixed with sodium chlorides, and carbon-based compounds in addition of some ironsulfide compounds. The sulfide peaks shown in the EDS spectra for M2 system might be related to the addition of ferrousammonium sulfate in the growth medium and the sulfide by-products formed by the survived SRB cells, as the Neem extractdid not inhibit the SRB growth completely.

However, the EDS spectra for M1 and M2 reveal substantial differences in the sulfide levels between the two systems. Theaverage sulfide level under M1 conditions was #27 wt.% while it was #3 wt.% for M2 systems, (Table 2). These observationssuggest the inhibitory effects of the Neem extract. The presence of di-potassium hydrogen orthophosphate, sodium chloridein the growth media might lead to the precipitation of phosphorous, sodium chloride on the surfaces exposed to M1 and M2systems [28].

The EDS elemental analysis for both systems (Table 2) suggests that the surface deposits and corrosion products in thepresence of the SRB consortium under M1 system is composed of significant accumulations of sulfur-based compounds.In comparison, the surface exposed to M2 solution is covered mainly with organic compounds in addition to iron oxides.There is a substantial amount of surface deposits and corrosion products growing upward Fig. 2A, observed for the M1system, which is most likely due to the biofilm matrix whose nature is polysaccharidic and viscoelastic [2,28]. Conversely,the corrosion products for the M2 system exhibit a completely different thin-flat layer with a hard texture partially coveredwith flaky cracked layer (Fig. 3A) suggesting minimum biofilm growth on the surface.

Higher magnification FE-SEM images display the nature of biofilm developed under M1 as shown in Fig. 4. The presenceof the SRB consortium together with the produced corrosion products has resulted in a heterogeneous morphology andthickness. The FESEM micrographs (Fig. 4) reveal the presence of the corrosion products, cells, spores and EPS fibersdistributed over the coupon. At the conclusion of the experiment, the steel substrate was covered with a porous black layer.A jelly-like substance was observed among the corrosion products, which was speculated to be biofilm-produced EPS. Thehigh density of sessile rod-shaped bacteria mixed with corrosion products and EPS was observed. EPS and corrosion productshave been reported to occupy 75–95% of biofilm volume, while 5–25% is occupied by the metabolizing cells. Reports haveshown that EPS and corrosion products occupy 75–95% of biofilm volume, while 5–25% is occupied by the sessile bacteria[29]. Higher concentrations of hydrogen sulfide, phosphate-based compounds and other biotically potential corrosion-influencing compounds are likely to be promoted by SRB metabolism and biofilm formation [3,6,28,30].

Conversely, the nature of corrosion film for the M2 system exhibits a completely different thin-flat layer with a hard tex-ture and small number of sessile bacteria as shown in Fig. 5. It can be seen that the density of sessile bacteria on the surface issubstantially less than those under M1 conditions. The small density of sessile bacteria revealed under M2 conditionsprovides evidence on the inhibitory influence of the Neem extract. The density of sessile bacteria is considered a significantfactor in MIC. It is important to note that sessile cells in a biofilm are considered the dominant contributor to MIC [31].

The corrosivity of the biofilm depends on different factors including sessile cell density and their enzyme activities [31].Both planktonic and sessile cells might produce sulfide. The sulfide produced by planktonic cells might diffuse to reach the

Table 2Comparative of EDS analysis corresponding to biotic system without Neem (M1) and abiotic system with Neem extracts (M2) systems, respectively.

Wt.% Element C O Na Si Fe S Cl P Mn

M1 systemLight region 18.69 ± 0.04 21.6 ± 0.02 0.92 ± 0.03 – 24.43 ± 0.35 26.59 ± 0.20 3.78 ± 0.11 4.00 ± 0.22 –Dark region 12.27 ± 0.05 31.26 ± 0.02 – – 31.90 ± 0.77 11.69 ± 0.12 5.69 ± 0.09 7.27 ± 0.11 –

M2 SystemBlack region 16.20 ± 0.03 11.34 ± 0.05 – – 49.19 ± 0.33 4.96 ± 0.17 3.15 ± 0.14 1.38 ± 0.07 3.61 ± 0.09White region 13.43 ± 0.02 21.53 ± 0.04 0.96 ± 0.13 1.04 ± 0.08 27.62 ± 0.19 – 10.48 ± 0.47 3.85 ± 0.10 –

96 S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103

metal surface causing hydrogen sulfide corrosion; however under normal conditions, the contribution from planktonic cellsto the local sulfide concentration at the metal surface is small compared to the locally produced sulfide by sessile cells, due todilution effects of the bulk fluid, corrosion products and biofilm structures [31]. This is supported by the fact that in theabsence of an SRB biofilm, pitting is not observed in the test samples [31].

(B) EDS analysis for the light region (A1) (C) EDS analysis for dark region (A2)

AAB C

Dark Region (A2) Light Region (A1)

Fig. 3. (A) FE-SEM images for API 5L X52 surface exposed to biotic system with Neem extract (M2) and EDS analysis for the light and dark regionrespectively.

Fig. 4. FE-SEM images for the biofilm developed on API 5L X52 surface exposed to biotic system with no Neem extract (M1).

Fig. 5. FE-SEM images for the biofilm developed on API 5L X52 surface exposed to biotic system with Neem extract (M2).

S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103 97

3.3. Morphology of surfaces after cleaning

Fig. 6 displays the FE-SEM images of the steel surface exposed to the M1 system after cleaning. As shown in Fig. 6, thesteel surface exhibits aggressive pitting colonies. In comparison, Fig. 7 shows the FE-SEM images of the steel surface exposedto the M2 system, which reveal minor pitting on the surface as the polishing marks are evident. These images suggested theinhibition effects of Neem extract under M2 conditions.

3.4. Corrosion mechanisms in presence of SRB-biofilm

There are different mechanisms by which anaerobic SRB can facilitate corrosion which have been suggested in literature[31,32]. Examples of mechanisms include cathodic depolarization, iron sulfide galvanic coupling and direct electron uptake[7,31–32]. The main electrochemical reaction involves the production of sulfide via SRB metabolic activities. SRB utilizecathodic hydrogen via hydrogenase enzyme to obtain the required electrons to reduce sulfate to sulfide by the followingreaction [1–3,32]:

SO2"4 þ 8Hþ þ 8e" ! HS" þ OH" þ 3H2O ð1Þ

In a deaerated environment, hydrogen is produced by the water dissociation cathodic reaction as shown below;

2H2Oþ 2e" ! H2 þ 2OH" ð2Þ

Electron transport reactions lead to proton motive force formation that supplies energy to the cells [3,26]. Somebiologically produced sulfide ions will convert to hydrogen sulfide especially at acidic pH as follows [32]:

HS" þHþ ¼ H2S ð3Þ

Fig. 6. FE-SEM images for the API 5L X52 clean surface exposed to biotic system with no Neem extract (M1).

Fig. 7. FE-SEM images for the API 5L X52 clean surface exposed to biotic system with Neem extract (M2).

98 S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103

The production of hydrogen sulfide and the oxidation of iron (anodic reaction) promotes the formation of iron sulfide asfollows [1–3,32]:

Fe! Fe2þ þ 2e" ð4Þ

Fe2þ þH2S! FeSþ 2Hþ ð5Þ

3Fe2þ þ 6OH" ! 3FeðOHÞ2 ð6Þ

Under M1 conditions, the SRB metabolic activities have enhanced the corrosion process through the depolarization ofhydrogen, production of sulfide and the formation of semi conductive iron sulfide film on the surface [31,32]. In comparison,under M2 conditions, the inhibition effect of the Neem extract might have eliminated the SRB metabolic activities. Moreover,a protective shielding layer might have developed under M2 conditions owing to the organic nature of the Neem extract. Thisprotection shield might provide further protection of the steel surface against the growth medium.

3.5. Open circuit potential (Ecorr)/polarization resistance (Rp) measurements

The variation of OCP as a function of time for the biotic system without Neem (M1) and biotic system containing Neemextract (M2) has been shown in Fig. 8. The OCP as a function of time revealed that in the biotic system (M1), a substantialshift of OCP towards noble values ("620 mV SCE"1) occurred for the first 100 h and then remained stable throughout theperiod of exposure. This potential shift might be attributed to the growth of the SRB species, their metabolic activitiesand biofilm development. SRB attached to the coupon surface, colonized and reproduced to form a biofilm, and the activitiesof microbes in this biofilm subsequently alter the electrochemical processes taking place at the steel surface [33]. Thesealterations might be driven by sulfide production, iron sulfide formation and even EPS production. These factors collectivelyenhance the reduction quality of the system and accelerated anodic dissolution [28,33].

On the other hand, the biotic system containing Neem extract (M2) remained more or less steady at approximately"640 mV SCE"1, except for the small initial increase. The initial increase might be attributed to the accumulation of Neemextract, biofilm and growth medium constituents such as potassium, sodium chloride and phosphorous on the couponsurface [28,33]. Comparing the OCP trend for the two systems, it can be noticed that the M2 system had nobler OCP valuescompared to the M1 system up to 100 h of immersion.

The positive shift in OCP is known as ennoblement and has been widely cited for different alloys exposed to microbes indifferent environments including fresh, brackish and seawaters [33]. The exact mechanism of ennoblement has not been re-solved. In many cases, ennoblement has been attributed to microbial colonization and biofilm formation, which collectivelyresult in organometallic catalysis and acidification of the electrode surface [2,33]. Noticeably, the OCP shift (ennoblement)observed under M1 conditions is correlated very well with the growth trend of SRB shown in Fig. 1. Dickinson et al. [34] hasrelated ennobled potential to cell density and biological activity in the biofilm by measuring ATP accumulation, electrontransport activity and lipopolysaccharide content [34]. The absence of ennoblement under M2 conditions supports theinhibition effect induced by the Neem extract.

Correspondingly, the linear polarization resistance (LPR) variation for M1 and M2 systems has been shown in Fig. 9A. TheRp as a function of time data revealed that in the biotic system with no Neem extract (M1), an initial increase to#2100 X cm2

0 50 100 150 200-710

-700

-690

-680

-670

-660

-650

-640

-630

-620

SRB SRB with Neem

Pote

ntia

l vs.

SCE

(mV)

Time (h)

Fig. 8. Open circuit potential (OCP) variations under biotic condition without Neem extracts (M1) and with 4% Neem extracts (M2).

S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103 99

at 24 h was observed followed by a substantial decrease to #200 X cm2 at around 100 h. After this time, Rp remained stablethroughout the period of exposure. The initial increase of the Rp values might be attributed to the formation of unstableprotective conditioning layer. This layer might be composed of a mixture of salts, iron sulfide, potassium and carbon-basedcompounds on the electrode surface. The following decrease in the Rp is attributed to the aggressiveness factors of thebiofilm and the active metabolisms of the sessile bacteria. The production of sulfide by SRB species, and subsequentaccumulation of biofilm, EPS and semi-conductive iron sulfide layers on the steel surface might have introduced multiplecathodic side reactions and promoted the corrosion process [6,31,32].

In contrast, the Neem extract containing biotic system (M2) showed higher Rp values over the system without Neem, thussuggesting an inhibition effect. The Rp as a function of time data revealed that in the biotic system with Neem extract (M2),an initial increase to #3500 X cm2 at 24 h was observed followed by a substantial decrease of Rp to 1100 X cm2 at 100 h.After this time, the Rp decreased and remained stable at #500 X cm2 throughout the period of exposure. The high initial in-crease of the Rp values might be related to the chemical adsorption of the phytochemical components of Neem on the surface[35]. This chemical adsorption process might result in the formation of unstable protective conditioning layer. The inhibitioneffect of Neem as seen from the Rp trend in Fig. 9A clearly indicates the antibacterial properties of the Neem extract.

Polarization resistance values can used to the determine corrosion current density, (icorr) using the following equation[36].

Icorr ¼ 1=Rp ( ðba ( bc=ð2:3ðba þ bcÞÞÞ ð7Þ

where ba and bc are the anodic and cathodic slopes, respectively.The corrosion rate plots over time for the M1 and M2 systems are shown in Fig. 9B. The average corrosion rate recorded

for the biotic system with no Neem (M1) was #35 mpy. In contrast, the average corrosion rate recorded for the biotic systemcontaining Neem extract (M2) was #14 mpy. These results revealed that Neem extract at 4% concentration has reduced thecorrosion rate by more than 50%.

3.6. Electrochemical impedance spectroscopy (EIS)

Fig. 10A and B show the EIS Zmod-frequency and phase angle-frequency Bode curves at selected time intervals for thebiotic system containing no Neem (M1). Both types of Bode curves show behavior characteristic of a corroding metal atlow time intervals which is revealed from the observed resistive response at low frequencies (horizontal Zmod curve andphase angles nearing 0"), confirming increased iron dissolution caused by bacterial activity. As the SRB colonize over thesteel surface and form a biofilm, there is a tendency for localized corrosion to facilitate, producing a noticeable drop in Zmodvalues at the low frequency end and a change in the appearance of phase angle Bode curves with time.

In contrast, Fig. 11A and B present the EIS Zmod-frequency and phase angle-frequency Bode curves at similar time inter-vals for the system containing Neem extract (M2). The Bode curves in this case are significantly different from those for theM1 system and there is no notable difference in the appearance of the shapes of the Bode curves with time as was seen incase of the M1 system, implying corrosion inhibition effects.

The impedance spectra for both systems M1 and M2 fitted a one time constant equivalent circuit model comprising of asolution resistance (Ru) in series with a parallel combination of polarization resistance of steel (Rp) and capacitance of thebiofilm (Yo) as shown in Fig. 12. The capacitance of the biofilm has been represented by a constant phase element (CPE) [37].The presence of CPE can be explained by dispersion effects that are caused by microscopic roughness of a surface or biofilm

0

500

1000

1500

2000

2500

3000

3500 SRB SRB with Neem

R p (Ω

cm2 )

Time (h)0 50 100 150 200 0 50 100 150 200

0

20

40

60

Corr

osio

n Ra

te (m

py)

Time (hours)

SRB SRB with Neem

Fig. 9. (A) Polarization resistance (Rp) and (B) Corrosion rate variations under biotic condition without Neem extracts (M1) and with 4% Neem extracts(M2).

100 S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103

malformation on the sample surface [38–40]. In case of a CPE, the value of alpha, heterogeneity parameter, is less than 1[25,28,41].

To summarize, surface analysis and electrochemical evaluation results suggest the inhibition effect of the Neem extractfor the SRB caused MIC of X80 linepipe steel. This statement is supported by the low planktonic cells density, low sulfideconcentrations, less sessile bacterial density, less pitting and low polarization resistance observed under M2 conditions com-pared to those under M1 conditions. The corrosion inhibition shown by the Neem extract might be explained by thefollowing:

Neem has significantly reduced the contribution of SRB in the corrosion process. Neem has minimized the growth of SRBcells, subsequently reduced the sulfide productions, sessile cell density and biofilm development.

It is proposed that Neem might have provided protection to the steel surface by forming an organic layer and shieldingthe surface for SRB colonization. Neem extracts are considered an incredible rich source of naturally synthesized chemical

10-2 10-1 100 101 102 103 104 105100

101

102

103Z m

od (Ω

cm2 )

Frequency (Hz)

2 h 48 h 96 h 144 h 192 h

10-2 10-1 100 101 102 103 104 10520

0

-20

-40

-60

-80

Phas

e An

gle

(°)

Frequency (Hz)

2 h 48 h 96 h 144 h 192 h

Fig. 10. EIS data for biotic condition without Neem extracts (A) Zmod-frequency Bode curve (B) Phase angle-frequency Bode curve.

10-2 10-1 100 101 102 103 104 105

101

102

103

Frequency (Hz)

2 h 48 h 96 h 144 h 192 h

10-2 10-1 100 101 102 103 104 10520

0

-20

-40

-60

-80

Phas

e An

gle

(°)

Frequency (Hz)

2 h 48 h 96 h 144 h 192 h

Z mod

(Ωcm

2 )

Fig. 11. EIS data under biotic condition with 4% Neem extracts (M2); (A) Zmod-frequency Bode curve (B) Phase angle-frequency Bode curve.

Fig. 12. Circuits model used to fit EIS data.

S.M. Bhola et al. / Engineering Failure Analysis 36 (2014) 92–103 101

compounds [35]. They contain large number of different organic chemical compounds. These compounds might have formedadsorbed intermediates (organo-metallic complexes) such as iron –polyethylene [Fe-PE], which can provide a protectiveshield to the metal surface. Okafor et al. [35] reported the inhibition effects of Azadirachta indica Neem extracts on mild steelcorrosion in H2SO4 solution [35]. Their investigation has revealed that Neem extracts functioned as good inhibitors in H2SO4

solutions due to the chemical adsorption of the phytochemical components of the plant on the metal surface.

4. Conclusions

This investigation provides preliminarily investigation on the effects of the Neem extract on the SRB induced biocorro-sion. The outcome of this investigation has produced promising results that show the inhibitory effects of 4 wt.% Neemextract on the corrosion influenced by SRB consortium. Neem is a renewable, biodegradable, non-toxic resource and is safefor the handling personnel in the plant facilities.

The results revealed that Neem extract at 4% concentration has reduced the corrosion rate by more than 50%. Obtaining50% reduction in SRB induced corrosion is significant as it will reduce the use of biocide in the industry. The present researchalso validates the use of Neem as an efficient biocide as it meets its requirements as mentioned above. Neem has significantlyreduced the contribution of SRB in the corrosion process by reducing the growth of SRB cells, subsequently reduced thesulfide productions, sessile cells density and biofilm development. Moreover, it is quite possible that Neem extract mighthave been adsorbed on the metal surface and provided an organic protective coating against the medium. More work isneeded to define the exact mechanism of Neem extract inhibition influence on MIC.

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