Surface Modification of Mild Steel with Thermally Cured Antibacterial

Article pubs.acs.org/IECR

Surface Modification of Mild Steel with Thermally Cured Antibacterial Poly(vinylbenzyl chloride)−Polyaniline Bilayers for Effective Protection against Sulfate Reducing Bacteria Induced Corrosion Li Lv,† Shaojun Yuan,*,† Yu Zheng,† Bin Liang,† and Simo O. Pehkonen‡ †

Multi-phase Mass Transfer & Reaction Engineering Lab, College of Chemical Engineering, Sichuan University, Chengdu 610065, China ‡ Department of Environmental Sciences, University of Eastern Finland, 70740 Kuopio, Finland S Supporting Information *

ABSTRACT: With the objective of developing anticorrosive conductive polymeric coatings to combat microbially induced corrosion (MIC), a facile and green synthesis approach based on a thermally induced reaction was described. Thermal curing of polyaniline (PANI) was carried out from the silanized mild steel (MS) surface containing reactive epoxy groups, followed by thermally induced N-alkylation of PANI by hydrophobic 4-vinylbenzyl chloride (VBzCl) to produce biocidal functionality. The so-synthesized MS coupons with hydrophobic poly(vinylbenzyl chloride) (PVBC)−quaternized PANI bilayer coatings were investigated for their anticorrosive and antibacterial properties toward biocorrosion induced by sulfate-reducing bacteria (SRB). Antibacterial assay results revealed an evident decrease in the bacterial attachment and the formation of biofilm. The QPANI− PVBC bilayer coatings showed a high corrosion resistance (inhibition efficiency >97%) and stability to resist SRB-induced corrosion. Thus, the QPANI−PVBC bilayer coated MS substrates can be used as effective polymeric coatings to protect the steelbased equipment in corrosive marine environments. more than 75% of the corrosion in oil fields and over 50% of all the microbial corrosion are caused by SRB.14 As a group of strict anaerobes, SRB can obtain their energy by oxidizing some organic compounds or molecular hydrogen (H2) while reducing sulfate (SO42−) to hydrogen sulfide (H2S).15 The main mechanisms proposed to interpret the SRB-induced corrosion include cathodic depolarization theory,16 local acid cell formation,17 creation of galvanic iron/iron sulfide cells,18 production of volatile phosphorus compounds,19 bacterial production of sulfides to induce anodic dissolution,17 and chelation activities of extracellular polymeric substances (EPS) to metallic ions.20 However, the real reason why SRB can induce the accelerated corrosion process is still unknown and incompletely understood, as several researchers have recently questioned the well-established cathodic depolarization theory on the cathodic electron uptake by hydrogen (H).21,22 They claimed direct electron consumption by iron rather than by hydrogen. In spite of much controversy, it becomes evident that both biogenic sulfide (H2S) and biofilm formation are crucial factors to aggravate the SRB-induced corrosion. It is therefore of great significance to simultaneously prevent aggressive sulfide anions and biofilms from attacking the metal substrates for the inhibition of corrosion induced by SRB. Different strategies have been proposed to inhibit the growth of SRB and their activities on the metallic substrrates for the

1. INTRODUCTION Due to its low price, its good malleability and machinability, and its steel material properites, mild (or carbon) steel has been utilized in a myriad of fields such as marine, chemical processing, petroleum, mining, and construction.1 However, the major drawback of mild steel is its limited resistance to corrosion under harsh environents. In particular, mild steel is susceptible to microbiologically influenced corrosion (MIC), or biocorrosion,2−5 which is directly associated with the undesirable colonization of indigenous bacteria on a metal surface to mediate accelerated metal dissolution.6,7 MIC has been a serious problem in various fields, as it is responsible for at least 20% of all damaging corrosion with a direct cost of $30−50 billion annually worldwide.8 MIC is a complicated corrosion process involved in a synergitic activies from bacteria, biofilms, metabolites, and aggressive ions in electrolytes.9 Microorganisms tend to adhere on a solid surface, colonize, proliferate, and excrete polysaccharides to protect themselves inside the biofilm matrix, which results in the localized gradients of corrosive anions of Cl− and S2−, pH, and dissolved oxygen, which is extremely corrosive to the underlying metals.10 Therefore, MIC is a localized type of corrosion in most cases, including crevice corrosion, pitting corrosion, and under deposit stress cracking, dealloying, etc.11 Various bacteria have been found to be involved in the MIC process, such as sulfate-reducing bacteria (SRB), manganese oxidizing bacterica, iron oxidizing/reducing bacteria, acidproducing bacteria, and slime-producing bacteria.12 Among them, sulfate-reducing bacteria have been extensively known as the main culprits of microbial corrosion.13 It is estimated that © 2014 American Chemical Society

Received: Revised: Accepted: Published: 12363

April 24, 2014 July 11, 2014 July 16, 2014 July 16, 2014 dx.doi.org/10.1021/ie501654b | Ind. Eng. Chem. Res. 2014, 53, 12363−12378

Industrial & Engineering Chemistry Research

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Figure 1. Schematic diagram illustrating the process of silanization of the hydroxylated mild steel (MS-OH), thermally curing PANI, thermally induced N-alkylation of the PANI layer.

poly(styrene-co-N-vinylpyrolidone),14 polyaniline,36 poly(4-vinylpyridine),37 poly(dimethylamino)ethyl methacrylate,38 poly(N-methylaniline),39 and poly(o-phenylenediamine),40 have been tailored onto metal substrates to inhibit MIC. Among them, conducting polymers attact much attention due to their anticorrosive and antimicrobial properties. The redox properties of conducting polymers can passivate metal substates to generate a protective oxide layer,41 while the positively charged nitrogen groups derived from the doped state of conducting polymers render the substrates with biocidal properties to inhibit the initial bacterial attachment and biofilm formation.42 However, only a few literature sources have reported on the utilization of conductive polymers as protective coatings for the inhibition of microbial corrosion induced by SRB.36,39 Polyaniline (PANI) is the first among conducting polymers to be used in practice, as an antistatic coating and corrosion inhibitor, due to its high chemical stability, nontoxicity, good processability, and stable intrinsic redox state derived from the imine groups of the PANI chain.43 PANI coatings can protect the underlying metallic substrates against corrosion by both anodic protection, which is caused by an oxide layer formed by the passivation reaction of PANI,44 and cathodic protection from the barrier ability of emeraldine (EM) base PANI coatings.45 Electrodeposition is usually used to coat conductive polymeric coatings on the metal substrates for corrosion inhibition.46 However, electrosynthesized PANI coatings are severely limited in practical usage by their poor adhesion. To address the adhesion problem of PANI coatings, the usage of organic acids instead of mineral acids as the electrolyte,47

mitigation of SRB-induced corrosion. As the most commonly used method, biocide treament is far from satisfactory due to the toxicity of biocides to the environment, the low effeciency against sessile microorganisms within the biofilm, and the high cost of dosing large amounts of biocides in an aquatic system.23 To minimize the environmental toxicity and enhance the biocidal functionality of biocides against biofilms, natural biocides, such as amino acids and natural extracts, have been used together with traditional biocides to synergistically act on the MIC of SRB.24−26 Organic inhibitors have gained considerable attention in inhibiting MIC in recent years,27−30 but they face similar dilemmas to biocides on their inherent toxicity to the environment, high cost, and the difficulty in implementing them in open systems. Alternatively, cathodic protection has been proven to be less effective to resist the SRB adhesion and the initiation of localized corrosion caused by SRB.31 On the other hand, protective antifouling paints, which are formulated with toxic copper or organotin compounds, have been completely banned from usage in marine environments since 2008 due to their adverse effect on nontargeted creatures.32 Based on the fact that the initial bacterial attachment and biofilm formation are the first step in initiating MIC, in recent years considerable efforts have been devoted to the development of green polymeric coatings incorporated with antimicrobial moieties. Some researchers have used antibacterial inorganic coatings, such as Ag nanoparticles,33 TiO2,34 CuO and ZnO,14 and graphene coatings,35 to protect the underlying metals from MIC. Alternatively, various polymeric coatings containg pendent or incorporated antimicrobial units, such as 12364

dx.doi.org/10.1021/ie501654b | Ind. Eng. Chem. Res. 2014, 53, 12363−12378


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predeposition of an adhesive primer layer,48 and doping PANI as an additive into matrix coatings49 have been adopted. In our previous study, an antibacterial poly(4-vinylaniline)−PANI bilayer coating was developed by combination of surfaceinitiated atom transfer radical polymerization and in situ chemical oxidative graft polymerization for MIC protection.36 The covalently tethered PANI chains not only preserved the intrinsic properties of the PANI homopolymer, but also displayed high performance to inhibit SRB-induced corrosion. However, these surface-grafting approaches require very strict reaction conditions, and the acid electrolyte for in situ chemical oxidative graft polymerization is somewhat corrosive to the underlying metallic substrates. Therefore, an alternative facile and green synthesis approach for depositing PANI coatings is highly desired. Herein, the main purpose of this study is to tailor the mild steel (MS) surface with antibacterial poly(vinylbenzyl chloride)−polyaniline (PVBC−PANI) bilayer coatings via thermally induced reactions to effectively inhibit SRB-induced corrosion in artificial seawater. As schematically shown in Figure 1, an epoxy-group-containing trimethoxylsilane was immobilized on the active mild steel (MS-OH) surface to provide anchoring sites for thermal curing of PANI. The thermally cured PANI in its EM base form was subsequently quaternized by thermally induced N-alkylation by vinylbenzyl chloride (VBzCl). The positively charged nitrogen (N+) compounds of the PANI, doped with the counter chloride ions (Cl−) formed during the alkylation, possessed a conductive state with a biocidal functionality. Concurrent with the alkylation reaction, the vinyl groups of VBzCl take part in the graft polymerization reactor to form a hydrophobic PVBC layer on the PANI surface.50 Each modification step was characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and static water contact angle measurements. The evaluation of bactericidal and corrosion behaviors of the sosynthesized coatings was performed in a simulated seawaterbased modified Barr’s (SSMB) medium in the presence of marine Desulfovibrio desulf uricans (D. desulf uricans) strains by antibacterial assays and electrochemical studies, respectively.

(defined as the pristine MS) coupons were cleaned and treated in an alkaline piranha solution to produce a hydroxylated (referred to as MS-OH) surface. The detailed procedures of the pristine MS and MS-OH coupon preparations are described in the Supporting Information. The silanization of the MS-OH surface was carried out to introduce active epoxy groups for subsequent thermal curing reaction. Briefly, the MS-OH coupons were immersed in 20 mL of a toluene solution containing 5 vol % GPTS for 24 h to immobilize a selfassembled silane monolayer. The resulting GPTS-immobilized (referred to as the MS-GPTS) coupons were washed with copious amounts of toluene, ethanol, and deionized water, respectively, and then dried in a vacuum oven at 50 °C for 2 h. The polyaniline (PANI) conductive polymers were synthesized via chemical oxidative polymerization using procedures described in detail in the Supporting Information. Thermally induced curing of the PANI was carried out to introduce the PANI layers on the MS-GPTS substrates using procedures similar to those described previously.55 About of 0.1 mL of a 20 mg/mL N-methyl-2-pyrrolidone (NMP) solution of emeraldine (EM) base PANI was introduced onto the MS-GPTS substrate surface. A thin liquid layer of EM base PANI can be formed evenly on the MS-GPTS surface by the spontaneous spreading of the fluid. The EM base solution coated surfaces were dried at 40 °C under atmospheric pressure for 6 h. Subsequently, the EM base coated surface was subjected to 1 M HCl for 2 h to protonate the EM base, followed by thermal treatment in a vacuum oven at 100 °C for 6 h. The thermal curing of physically adsorbed EM salt was achieved by chemical reaction between the imine groups of PANI and the epoxy groups on the Cu-GTPS surface. Finally, the thermally cured PANI layer in its EM salt form was immersed in a 0.5 M NaOH for 2 h to convert to the neutral EM base form.The resultant PANIcoated MS substrates were defined as the MS-c-PANI surfaces. 2.3. Thermally Induced N-Alkylation of the MS-c-PANI Surface. The MS-c-PANI surfaces with the thermally cured PANI layer in its neutral EM base form were quaternized via thermally induced N-alkylation to generate a biocidal functionality, using procedures similar to those described previously.56 The MS-c-PANI coupons were treated in a 20 vol % dioxane solution of 4-vinylbenzyl chloride (VBzCl) at 80 °C for 8 h. The resulting MS surface with PVBC quaternizedPANI bilayer coatings was defined as the MS-c-QPANI−PVBC surface. After the N-alkylation, the MS-c-QPANI−PVBC substrates were washed with a large amount of chloroform and deionized water to remove the unreacted VBzCl monomer, respectively, and then dried at 50 °C under dynamic vacuum overnight prior to further characterization. The color of the PANI-coated MS surface was changed from dark blue of the EM base to dark green of the quaternized EM moieties. 2.4. Surface Characterization. Each modification step of the MS substrates was characterized by X-ray photoelectron spectroscopy (XPS). The XPS measurements were performed on a Kratos AXIS HSi spectrometer using a monochromatic Al Kα X-ray source (1486.6 eV photons) such as those described in detail previously.51 The static water contact angle of each substrate surface was measured using the sessile drop method at ambient temperature with a 3 μL water droplet and a telescopic goniometer (Rame-Hart, Inc., Mountain Lake, NJ). The averaged contact angles were obtained from at least four locations on each substrate. The thickness of the polymer coatings was obtained by the ellipsometric measurement on a variable angle spectroscopic ellipsometer (Model VASE, J. A.

2. EXPERIMENTAL SECTION 2.1. Materials. A 3-mm-thick AISI C1020 mild steel sheet (nominal composition (%): C 0.17−0.23, P 0.04, Mn 0.3−0.6, S 0.05, and Fe balance) was obtained from Metal Samples Co. (Munford, AL). Chemicals, such as aniline (99.5%), 4vinylbenzyl chloride (90%, VBzCl), (3-glycidoxypropyl)trimethoxylsilane (GPTS, 98%), ammonium persulfate (98%), a H2O2 solution (30%), and solvents, such as N-methyl-2pyrrolidone (NMP, 99.5%), ethanol (anhydrous, 99.5%), acetone, chloroform, toluene (99.8%), and 1,4-dioxane (anhydrous, 99.8%), were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and were used as received. Culture medium components such as yeast extract were obtained from Oxoid (Hampshire, U.K.). A Gram-negative marine anaerobic strain, D. desulf uricans (ATCC 27774), was provided by the American Type Culture Collection. The LIVE/ DEAD staining kit was obtained from Molecular Probes, Inc. (BacLight, Eugene, OR). The freshly prepared phosphate buffered solution (PBS, containing NaH2PO4 of 4.68 g/L and Na2HPO4 of 8.662 g/L) was adjusted to a pH value of 7.4 prior to being sterilized in an autoclave prior to use. 2.2. Silanization of MS-OH Surfaces and Thermal Curing of EM Salt of PANI. The newly polished mild steel 12365



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predetermined 3, 10, and 21 days of exposure time, the MS coupon was removed and mounted into a specific PVDF holder, leaving a coupon area of 0.785 cm2 contact with inoculated medium, to serve as the working electrode. The steel coupon embedded in the holder was connected with the working electrode using a round copper rod to remain conductive. A inert platinum rod was chosen as the counter electrode, and an Ag/AgCl/KCl (3 mol·L−1) electrode was chosen as the reference electrode. All of the electrochemical measurements were conducted on an Autolab PGSTAT30 (Metrohm, Utrecht, Netherlands). The measurement of Tafel plots were performed using a scan rate of 1 mV·s−1 in the range −250 to +250 mV relative to the open circuit potential (OCP), to determine the corrosion current densities (jcorr) and corrosion potentials (Ecorr). EIS data were recorded at the OCP in the frequency range 100 000−0.005 Hz using a 10 mV amplitude sinusoidal signal. The inhibition efficiency (η) of the conductive polymeric coatings can be calculated using the following equation:

Woollam, Lincoln, NE). The incident angles were chosen at 70 and 75° in the wavelength range 250−1000 nm using previous procedures.36 The average film thickness was determined from at least three different locations by processing the WVASE32 software package. The change in surface morphology of MS coupons after each surface modification step was characterized by scanning electron microscopy (JEOL JSM-5600, Tokyo, Japan). 2.5. Bacterial Adhesion and Viability Assays of the Surface-Functionalized MS Surfaces. Detailed procedures for the cultivation and inoculation of D. desulf uricans and the preparation of the SSMB medium are described in the Supporting Information. The biocidal functionality of surfacefunctionalized MS substrates was characterized by the bacterial adhesion and viability assays, respectively.36,38 SEM images were captured to evaluate bacterial adhesion profiles on the pristine MS and surface-functionalized MS substrate surfaces. Briefly, the pristine MS and surface-functionalized MS coupons were immersed in the D. desulf uricans inoculated SSMB medium with a cell density of 106 MPN·mL−1 for 3, 10, and 21 days. The coupon incubation was performed in a Don Whitley anaerobic chamber (Model MASC MG 50, Maharashtra, India) under an mixture gas of 5% H2, 5% CO2, and 90% N2 atmosphere. After the predetermined incubation time, the MS coupons were washed with a large amount of a sterilized PBS solution to get rid of the dead and poorly adherent bacterial cells, followed by immersion in a PBS solution containing 2.5 vol % glutaraldehyde (GA) for fixation at 4 °C for 6 h, and then they were stepwise immersed in 25, 50, 75, 90, and 100 vol % ethanol solutions each for 5 min for dehydration. The bacteria-attached coupons were finally dried in a vacuum desiccator prior to SEM imaging. To evaluate the viability of the bacteria adhered on the substrates, fluorescence microscopy (FM) images, stained with a mixture of SYTO 9 green fluorescent nucleic acid dye and propidium iodide (PI) red fluorescent nucleic acid dye, were obtained on the various substrates. As a membrane permeable dye, the SYTO 9 can stain both live and dead bacterial cells. PI can only stain the nonviable cells due to the rejection by cell membrane pumps. Thus, live and dead bacterial cells, which appear with green and red fluorescence, can be distinguished under the fluorescence microscope. At the end of the predetermined incubation periods, the MS coupons were stained by a 0.1 mL aliquot of a solution of the LIVE/DEAD Baclight kit on the substrate surface for 15 min. The FM images of stained coupons were visualized under a green filter (excitation/emission wavelengths, 420−480 nm/520−580 nm) or a red filter (excitation/emission wavelengths, 480− 550 nm/590−800 nm) on a Leica DMLM microscope. To quantitatively assess the biocidal functionality of the surfacemodified MS coupons, the viable cell numbers on each substrate surface were determined using the three-tube most probable number (MPN) method as a function of exposure time, using similar procedures reported previously.52 2.6. Anticorrosive Behavior of the Functionalized MS Coupons. Tafel plots and electrochemical impedance spectroscopy (EIS) data were obtained to determine the anticorrosive capability of the PANI coatings on the MS substrates. The pristine and surface-modified MS coupons, hung on nylon strings, were statically immersed in a 500 mL Wheaton bottle containing 400 mL of a simulated seawaterbased modified Barr’s (SSMB) medium, as shown schematically in Figure S1 (Supporting Information). At the end of

η% =

jo − jcorr jo

(1)

where jo and jcorr represent the respective corrosion current densities of the pristine MS and surface-modified MS coupons, both of which were obtained by analyzing Tafel plots. Subsequently, the corrosion rate (ν) was calculated by the following equation: ν=

jcorr KM w ndA

(2)

where K is a constant that defines the units for the corrosion rate, and the parameters of A, d, Mw, and n correspond to the surface area of coupons, density, atomic weight, and electron number of the MS coupons, respectively.

3. RESULTS AND DISCUSSION The synthesis of poly(vinylbenzyl chloride)−polyanline (PVBC−PANI) bilayer coatings on the mild steel (MS) surfaces via thermally induced reactions consists of a threestep reaction sequence, as schematically illustrated in Figure 1. Silanization of the hydroxyl-enriched MS (MS-OH) surfaces was first performed using trimethoxylsilane to introduce reactive epoxy groups as anchor sites for thermal curing (i.e., the MS-CPTS surface). Thermal curing of PANI was carried out to deposit the emeraldine (EM) base PANI on the MS substrates by the chemical reaction between the epoxy groups and amine groups (−NH−) of the PANI chain (i.e., the MS-cPANI surface). Thermally induced N-alkylation of PANI layers was achieved by the reaction between vinylbenzyl chloride (VBzCl) and the imine groups of the PANI chains to generate biocidal functionality. Thermally induced polymerization of VBzCl via vinyl groups took place simultaneously to form a hydrophobic PVBC layer (i.e., the MS-c-PANI−PVBC surface). Details of each modification step are discussed below. 3.1. Silanization of MS-OH and Thermal Curing of PANI. The pretreatment of the MS surface with a strong oxidizer, alkaline piranha solution (NH3·H2O:H2O2, 3:1, v:v), was performed to achieve the hydroxylated MS (i.e., MS-OH) surface.53 The MS-OH surface containing abundant hydroxyl groups (−OH) can be deduced from the decrease in the static water contact angle from about 55 ± 4° to about 26 ± 2° (Table 1). The surface chemistry profile of the MS-OH surface 12366



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adventitious adsorption of hydrocarbons during sample handling. This result is consistent with the appearance of a additional minor OC−O− peak component with BE at 288.6 eV (Figure 2b). The only Si−O peak component with BE at 101.5 eV in the Si 2p core-level XPS spectrum54 further confirms the formation of the GPTS monolayer on the MS surface through strong Si−O bonds. A thermal curing reaction provides an alternative approach to achieve strong adhesion of polymeric coatings onto a solid substrate surface.55 In this study, the PANI layers in their EM salt form were thermally cured at 100 °C for 6 h to induce reactions between the amine groups of PANI chains and the epoxy groups on the MS-GPTS surfaces. The thermally cured PANI layer was further converted from its EM salt form to the neutral EM base form (i.e., the MS-c-PANI surface). The water contact angle of the MS-c-PANI surface is about 48 ± 2°, and the thickness of the thermally cured PANI layer is around 2132 ± 43 nm (Table 1). Figure 2d,e shows the respective wide scan, C 1s, and N 1s core-level XPS spectra of the MS-c-PANI surface. In comparison that of the MS-GPTS surface, the signals of Si 2p, O 1s, and Fe 2p disappear in the wide scan XPS spectrum of the MS-c-PANI surface, indicative of the coverage of dense PANI layers on the MS substrates (Figure 2d). The [N]/[C] ratio of around 0.16 is consistent with the theoretical value of 0.167 of the aniline molecular structure. The C 1s corelevel XPS spectrum is curve-fitted into three peak components of C−H (284.6 eV), C−N (285.5), and C−N+ (286.2 eV) (Figure 2e).54 The C−N+ species is probably associated with the persistence of minor protonated PANI after the base treatment. The N 1s core-level spectrum of the MS-c-PANI surface is curve-fitted into peak components with BEs of 398.2 and 399.4 eV, attributable to quinonoid imine (N−) and benzenoid amine (−NH−), respectively (Figure 2f). The approximately equal proportion of the quinonoid imine and benzenoid amine is consistent with the 50% intrinsically oxidized EM base form of PANI.56 The residual high BE tail (>400 eV) in the N 1s core-level spectrum indicates that the minor positively charged nitrogen (N+) (i.e., protonated PANI) persists on the surface after the alkaline treatment, since the protonation and deprotonation of PANI is a dynamic equilibrium process.57 The [N+]/[NTotal] ratio is about 2.3%. Thus, the neutral EM base form PANI layers were thermally cured on the MS surfaces for the subsequent antibacterial assays and anticorrosion analyses. 3.2. Thermally Induced N-Alkylation of PANI Layers on the MS-c-PANI Surface. Ultraviolet (UV) or thermally induced N-alkylation with VBzCl has been used to dope PANI (in the EM base form) to maintain its conductive state and simultaneously form a hydrophobic polymer layer (PVBC) to act as the protective barrier for the undoping of the PANI layers.50 Thermally induced N-alkylation of the EM base PANI layer was accomplished by thermally induced graft copolymerization of VBzCl over the MS-c-PANI surface. Figure 2g,h shows the respective C 1s and N 1s core-level XPS spectra of the MS-c-QPANI−PVBC surface. The curvefitted N 1s core-level XPS spectrum is mainly composed of benzenoid amine (−NH−, BE of 399.4 eV) and positively charged nitrogen (N+, BE of 400.8 eV) species,54 respectively (Figure 2h). As compared to that of the MS-c-PANI surface, the disappearance of quinonoid imine (N−) and the proportional increase of positively charged nitrogen (N+) after the N-alkylation reaction, as well as the almost unchanged intensity of the benzenoid amine (−NH−), suggests that the

Table 1. Static Water Contact Angles of Different MS Substrate Surfaces

a

Pristine MS refers to a newly polished mild steel (MS) coupon. bMSOH was obtained after the pristine MS coupon was immersed in a alkaline piranha solution (NH3OH:30% H2O2, 3:1, v:v) for 30 min. c MS-GPTS was obtained after the MS-OH was immersed in a anhydrous ethanol solution containing 3-glycidoxypropyl methyldiethoxysilane (GPTS) for 24 h at room temperature. dMS-c-PANI was obtained by thermally curing EM salt PANI in a vacuum oven at 100 °C for 6 h. The EM salt form of PANI was converted to the neutral EM base after NaOH treatment. eMS-c-QPANI−PVBC surface was achieved by thermally induced N-alkylation of PANI in a 20 vol % dioxane solution of vinylbenzyl chloride (VBzCl) at 80 °C for 8 h. g The film thickness was obtained by a variable angle spectroscopic ellipsometer. hSD denotes standard deviation. iWCA refers to water contact angles in deionized water, determined by averaging the contact angles from at least three surface locations. The values in parentheses correspond to static water contact angles of various coupons in artificial seawater.

was determined by XPS, and the results are shown in Figure S2 (Supporting Information). The dominant O 1s signal in the wide scan spectrum (Supporting Information, Figure S2a) and the strong peak component with a binding energy (BE) at 531.7 eV in the curve-fitted O 1s XPS spectrum (Supporting Information, Figure S2c), attributable to hydroxide species,54 suggest a large amount of hydroxyl groups generated on the MS-OH surfaces. The curve-fitted O 1s core-level spectrum consists of three peak components with BEs at 530.1, 532.1, and 533.2 eV, attributable to oxide, hydroxide, and water,54 respectively, indicative of the formation of a thin oxide and hydroxide film on the MS substrate surface (Supporting Information, Figure S2c). Thus, a hydroxyl-enriched MS surface is generated upon treatment with the alkaline piranha solution. The introduction of epoxy groups as anchor sites onto the substrate surfaces was achieved by coupling of the MS surface with (3-glycidoxypropyl)trimethoxylsilane (GPTS). The presence of epoxy groups on the silanized MS surface leads to the increase in water contact angle to around 52 ± 3° (Table 1). The thickness of the GPTS layer is around 1.5 ± 0.4 nm (Table 1), indicative of the immobilization of a silane monolayer on the MS-OH surface. Parts a, b, and c of Figure 2 show the wide scan, C 1s, and Si 2p core-level spectra of the MS-GPTS surfaces, respectively. As compared to the wide scan XPS spectrum of the MS-OH surface, two additional Si 2p (at BE of about 99 eV) and Si 2s (at BE of about 151 eV) signals and the evident increase in relative intensity of the C 1s spectral line indicates the successful immobilization of GPTS on the MS surfaces (Figure 2a). The [Si]/[C] ratio, as determined from the Si 2p and C 1s core-level spectral area ratio, is about 0.12, which is comparable to the theoretical value of 0.167 for the GPTS structure. The slight deviation is probably caused by the 12367



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Figure 2. (a) Wide scan, (b) C 1s, and (c) Si 2p core-level spectra of the MS-GPTS surface; (d) wide scan, (e) C 1s, and (f) N 1s core-level spectra of the MS-c-PANI (protonated) surface; (g) C 1s, (h) N 1s, and (i) Cl 2p spectra of the MS-c-QPANI−PVBC surface.

(Figure 2i), indicative of the presence of PVBC layers on the MS-c-PANI surface. The occurrence of copolymerization of PVBC during the alkylation reaction is consistent with the notable increase in the surface hydrophobicity, as the water contact angle increases to about 85 ± 4° for the MS-c-QPANI− PVBC surface (Table 1). The thickness of the PVBC− quaternized PANI bilayer is ca. 2143 ± 32 nm (Table 1). The so-synthesized MS coupons are subsequently evaluated for their antimicrobial and anticorrosive behavior in a SRBinoculated artificial seawater. 3.3. Characterization of Surface Morphology. The change in surface morphology of the MS substrates after each functionalization step was characterized by SEM imaging. Figure 3 shows the top view and the cross-sectional view of SEM images for the pristine MS, MS-GPTS, MS-c-PANI, and MS-c-QPANI−PVBC surfaces. The pristine MS surface exhibits a relatively rough surface with evident scratches caused by the polishing process with abrasive papers (Figure 3a). After the alkaline piranha solution pretreatment and the GPTS silanization, the coverage of a silane monolayer seems to slightly decrease the surface roughness (Figure 3b), although the polishing scratches are still visible on the MS-GPTS surface. It is interesing to observe that the deposition of dense and compact thermally cured PANI layers results in a fairly smooth and homogeneous MS surface (Figure 3c), which indicates a uniform thermal-curing process of PANI over the silanized MS substrates. The thickness of the thermally cured PANI layers is

alkylation reaction takes place preferentially on the quinonoid imine (N−) rather than on the benzenoid amine (−NH−) of the PANI in EM base form. It has been reported previously that the quinonoid imine nitrogen is more reactive than the benzenoid amine nitrogen as a nucelophile during the Nalkylation reaction process.58 The alkylation ratio of the fully reduced PANI (i.e., leucoemeraldine or LM) was found to be less than 10%, but the EM base form PANI can achieve a degree of alkylation as high as 30%.58 The area ratio of [N+]/ [NTotal] is around 27.4% after the thermally induced Nalkylation with VBzCl, indicative of a high degree of Nalkylation reaction for the EM base PANI on the MS-cQPANI−PVBC surface. Figure 2i shows the Cl 2p core-level XPS spectrum of the MS-c-QPANI−PVBC surface. The curve-fitted Cl 2p core-level spectrum consist of two spin−orbit split doublets with BEs for the Cl 2p3/2 peaks at 197.2 and 200.3 eV, attributable to the ionic chlorine (Cl−) and the covalent chlorine (−Cl),54 respectively. The ionic chlorine (Cl−) derives from the aliphatic nucleophilic substitution reaction for the conversion of imine (N−) to the positively charged nitrogen (N+) with VBzCl. The ionic chlorine (Cl−) can serve as a counterion to the N+ components of PANI, resulting in the doping of PANI. The covalent chlorine (−Cl) is probably associated with the PVBC layers formed on the PANI-coated surface in concurrence with the alkylation reaction. The [Cl]/[C] ratio, as determined by the Cl 2p and C 1s core-level spectral area ratio, is about 0.049 12368



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Figure 3. SEM images to display surface morphologies of (a) pristine MS, (b) MS-GPTS, (c−e) MS-c-PANI, and (f, g) MS-c-QPANI−PVBC surfaces. (e) and (g) correspond to the cross-sectional SEM images of the thermally cured PANI layer and QPANI−PVBC bilayers, respectively.

about 2 μm from the cross-sectional SEM images (Figure 3d). This result is consistent with that obtained by the ellipsometry measurement. The edge profile of the SEM images further confirms the presence of dense PANI layers on the MS-c-PANI surfaces (Figure 3e). Upon the thermally induced N-alkylation and the accompanying copolymerization of PVBC on the PANI-coated MS surfaces, no significant change in the surface morphology can be observed, although the MS-c-QPANI− PVBC surface displays a slight increase in the surface roughness (Figure 3f,g).

3.4. Antibacterial Properties of the Surface-Functionalized MS Surface. 3.4.1. Bacterial Adhesion Assays. The bacterial adhesion profile on the pristine (i.e., newly polished) and functionalized MS surfaces was determined by SEM imaging after 3 and 21 days in a D. desulf uricans suspension of 106 MPN·mL−1. Figure 4 shows the representative SEM images of the pristine MS coupons after exposure to the sterile and the D. desulf uricans inoculated SSMB medium for 3 and 21 days. The MS surface displays no significant change in morphology after exposure to the sterile SSMB medium for 3 days (Figure 12369



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Figure 4. Representative SEM images of pristine MS surfaces after 3 and 21 days of exposure to (a, b) sterile SSMB medium and (c, d) D. desulfuricans inoculated SSMB medium. (e) and (f) denote the respective high-magnification (×3000) SEM images of the SRB cell-deposited MS surfaces after 3 and 21 days of exposure. (g, h) SEM images of the 3- and 21-day-exposed MS surface in the presence of D. desulf uricans upon the removal of bacterial cells and corrosion products.

thick and lumpy corrosion products and deposits have covered the entire the MS coupon surface (Figure 4d). The SEM image with a large magnification reveals the presence of the corrosion products (mainly iron sulfides), bacterial cells, and EPS in the biofilm of D. desulf uricans (Figure 4f). It has been reported that the biofilm contains 75−95% EPS and corrosion products and 5−25% metabolizing cells.11 Another notable feature is the heterogeneous morphology and the thickness of the biofilm over the MS substrates. The heterogeneity of the biofilm was found to faciliate the creation of localized gradients of aggressive anions, such as Cl− and S2−, thus triggering the

4a). Upon prolonging the exposure period to 21 days, discrete deposits and corrosion products formed on the MS surface and can be clearly observed (Figure 4b). The corrosion products in a salt crystal deposit form are reported to be composed of iron oxides mixed with sodium chloride, calcium, and carbon-based compounds that accumulated from the growth medium.59 Numerous small-clustered or microcolony bacterial cells are distributed over the pristine MS surface after exposure to the D. desulf uricans inoculated medium for 3 days (Figure 4c). The D. desulf uricans cells are observed to be rod-shaped and aggregate to form a patchy biofilm (Figure 4e). After 21 days of exposure, 12370



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Figure 5. Representative SEM images of (a−c) MS-c-PANI and (d−f) MS-c-QPANI−PVBC surfaces after 3 and 21 days of exposure to D. desulfuricans inoculated SSMB medium. (c) and (f) correspond to high-magnification (×3000) SEM images of SRB-attached MS-c-PANI and MS-cQPANI−PVBC surfaces after 21 days of exposure, respectively.

in microcolonies or in patchy biofilms, are distributed over the MS-c-PANI surface (Figure 5b), indicating that the PANI layers possess a limited biocidal functionality. The normal shape of the attached bacterial cells observed in the magnified SEM images further confirms the weak antibacterial capability of the surface to resist bacterial attachment and growth (Figure 5c). On the contrary, the MS-c-QPANI−PVBC surfaces exhibit poor bacterial adhesion throughout the exposure periods, as compared to the pristine MS and MS-c-PANI surfaces. Almost no bacterial cells can be observed on the 3-day-exposed surface of the MS-c-QPANI−PVBC substrates (Figure 5d). Moreover, only some single cells are sparsely distributed over the 21-dayexposed MS-c-QPANI−PVBC surface (Figure 5e), and the bacterial cells are oval- or rod-shaped with a reduced length of 2 μm or less (Figure 5f). These results suggest that the MS-cQPANI−PVBC surface possesses a high antibacterial property to inhibit the bacterial adhesion and biofilm formation. 3.4.2. Bacterial Viability Assays. The viability of adhered cells on the functionalized MS surfaces was determined by LIVE/DEAD fluorescence imaging. The distribution of viable and dead bacterial cells on various MS substrates was observed

initiation of various types of localized corrosion, such as pitting or crevice corrosion.60 The corrosion degree of the MS surface underlying the biofilm was characterized by the removal of the biofilm using sterile cotton by the procedures reported previously.51 Some shallow micropits or metastable pits are spotted and distributed over the MS surface upon the biofilm removal after 3 days of exposure (Figure 4g). Numerous deep micropits and macropits are clearly observed on the MS surface after 21 days of exposure upon removal of the corrosion products (Figure 4h). These results further confirm the previous finding that the steel-based substrates are susceptible to localized corrosion in the presence of SRB.4,9,61 Figure 5 shows the representative SEM images of the MS-cPANI and the MS-c-QPANI−PVBC surfaces after exposure to the inoculated medium for 3 and 21 days. Only a small number of individual bacterial cells are visible over the MS-c-PANI substrates after the initial 3 days of exposure (Figure 5a). The reduction of bacterial adhesion is ascribed to the antimicrobial properties of the EM base form PANI, which is derived from the positively charged nitrogen (N+) of the PANI chains.42 However, after 21 days of exposure, many bacterial cells, either 12371



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Figure 6. Representative fluorescence images of (a−d) MS-c-PANI and (e−h) MS-c-QPANI−PVBC surfaces under the green filter (a, c, e, g) and the red filter (b, d, f, h) after 3 and 21 days of exposure to D. desulfuricans inoculated SSMB medium. Scale bars: 50 μm.

with fluorescent microscope (FM) images, of which green fluorescence denotes live cells while red fluorescence represents dead cells. The FM images of the pristine MS surfaces are shown in the Supporting Information, Figure S3. Most of the attached bacterial cells on the pristine MS surface exhibit green fluorescent color (Supporting Information, Figure S3a,c), implying that the bacterial cells are viable cells with intact cell membrane. It is noted that the presence of numerous redstained dead cells on the 21-day-exposed pristine MS surface (Supporting Information, Figure S3d) are ascribed to natural apoptosis rather than the antibacterial activity in the growth process of D. desulf uricans. These results are consistent with the fact that that the steel-based substrate surfaces are favorable templates for the adhesion, growth, and proliferation of

microorganisms, and that the biofilm will form readily on such material in contact with bacteria. Figure 6 shows the FM images of the MS-c-PANI and the MS-c-QPANI−PVBC surfaces after exposure in the inoculated medium for 3 and 21 days. Only a few stained-green viable cells are visible on the MS-c-PANI surface during the initial 3 days (Figure 6a), which is significantly lower than those on the pristine MS surface. The result is in good agreement with the SEM observation that the EM base PANI possesses an antimicrobial activity. The antibacterial ability of PANI probably involves the following: (i) the positively charged nitrogen (N+) of doped (i.e., protonated) molecular PANI chains may result in the death of bacterial cells57 and (ii) the electrostatic interactions between the PANI molecules and the oppositely charged cell walls lead to the leakage of bacterial 12372



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contents.42 However, significantly more viable cells of D. desulf uricans (i.e., stained green) are present on the MS-c-PANI surface after 21 days of exposure, and result in the formation of microcolonies or large-sized colonies of bacterial cells (Figure 6c), indicating that the weak antibacterial activity of PANI cannot effectively resist the bacterial attachment, proliferation, and biofilm formation over extended time periods. In contrast, only some individual stained-green viable cells are present on the MS-c-QPANI−PVBC surface throughout the exposure periods (Figure 6e,g), especially that almost no viable cells are visible during the initial 3 days of exposure (Figure 6f). The above results indicate that hydrophobic quaternary ammonium moieties in the quaternized PANI chains possess high killing ability to the bacterial cells upon contact. It has been reported that the hydrophobic PVBC− quaternized PANI polymers possess a much higher antibacterial capability due to the long hydrophobic PVBC chains.62 Many researchers believe that the antibacterial activities of small molecular weight quaternary salts are regulated by a long hydrophobic chain, which can react with cytoplasmic membranes of cells, and that the antibacterial activity increases with an increase in the length of the substituted alkyl chain. Thus, the high antibacterial efficiency of the hydrophobic PVBC−quaternized PANI bilayer coating is ascertained. To quantitatively evaluate the antibacterial efficiency of the PANI-deposited MS surface, the attached viable cells were enumerated using procedures similar to those described previously.38 Figure 7 shows the number of viable cells adhered

hydrophobic alkyl chain of PVBC can effectively eradicate the D. desulf uricans cells in contact. 3.5. Anticorrosion Behavior of the Surface-Functionalized MS Coupons. 3.5.1. Tafel Plots. As a commonly used technique to monitor the instantaneous corrosion rate of a metal (or an alloy) substrate, Tafel plots are suitable to detect subprocess change at the metal/bacteria (or inhibitors, biocides, coatings) interface.60 Figure 8 shows the respective Tafel plots of the pristine MS and functionalized MS coupons for various exposure times in the sterile and the D. desulf uricans inoculated SSMB medium. The Tafel plots were quantitatively analyzed using GPTS 4.9 software to obtain the corrosion current density (jcorr), corrosion potential (Ecorr), and corrosion rate (ν). The inhibition efficiency (η) and the corrosion rate (ν) of the PANI-deposited MS coupons were calculated according to eqs 1 and 2, respectively, as shown in the Experimental Section. The analysis results of the Tafel plots results are summarized in the Supporting Information, Table S1. The corrosion potential, Ecorr, of the pristine MS coupons undergoes a slight increase in values with exposure time in the sterile SSMB medium from about −0.56 V after 3 days of exposure to about −0.49 V after 21 days of exposure (Figure 8a), while it shifts rapidly to a more negative value with exposure time in the D. desulf uricans inoculated SSMB medium (Figure 8b), and the values decrease to as low as about −0.74 after 21 days of exposure (Supporting Information, Table S1). As a widely recognized phenomenon, the decrease in the Ecorr values in the presence of SRB is probably associated with the enhanced anodic dissolution according to the mixed potential theory.63 The Ecorr values of the surface-modified MS coupons undergo a noticeable increase as compared to those of the corresponding pristine MS coupons (Figure 8c,d). In particular, the Ecorr values of MS-c-QPANI−PVBC coupons increase by at least 0.35 V relative to the pristine MS coupons throughout (Supporting Information, Table S1). The positive shift in Ecorr values is a common phenomenon for the polymeric coatings on the bare metal coupons. This phenomenon is probably caused by the barrier properties of polymeric coatings, which mainly result in anodic protection in terms of the mixed potential theory.64 The jcorr values of the pristine MS coupons gradually decrease with exposure time in the sterile SSMB medium due to the formation of the conditioning layers (mainly yeast extract) and oxide films. The jcorr values of the pristine MS coupons decrease rapidly to about 50.9 μA·cm−2 during the initial 10 days of exposure, and subsequently reduce to about 32.4 μA·cm−2 after 21 days of exposure in the D. desulf uricans inoculated SSMB medium (Supporting Information, Table S1). The reduction in the jcorr values is ascribed to the thick corrosion products (including biofilm and iron sulfides) formed on the MS substrates as observed from the SEM images (Figure 4d). As a result, the corrosion rate of the pristine MS coupons remains above 0.35 mm·year−1 in the SRB-containing artificial seawater, which is around 10 times higher than the industrial standard rate of less than 0.03−0.06 mm·year−1 for steel-based structures in marine environments.65 The jcorr values of the MS-c-PANI coupons are significantly reduced by more than 20-fold (Supporting Information, Table S1) compared to those of the pristine MS coupons during the initial 10 days of exposure, indicating that the thermally cured PANI coatings render the desired high corrosion resistance to the synergistic attack of SRB and aggressive Cl− and S2− anions. More pronounced decreases in jcorr values are observed on the MS-c-QPANI−

Figure 7. Number of viable cells attached to pristine MS, MS-c-PANI, and MS-c-QPANI−PVBC surfaces as a function of exposure time in D. desulfuricans inoculated SSMB medium.

on the pristine MS, MS-c-PANI, and MS-c-QPANI−PVBC surfaces after 3, 10, and 21 days of exposure in the D. desulf uricans inoculated SSMB medium. The viable cell counts on the pristine MS surface appears to increase rapidly from about 105 MPN·cm−2 after 3 days of incubation to more than 107 MPN·cm−2 after 21 days of exposure. The number of viable cells on the MS-c-PANI surface decreases by about 2 orders of magnitude relative to the pristine MS surface, although with a noticeable increase to around 105 MPN·cm−2 after 21 days of exposure. For the MS-c-QPANI−PVBC surface, the lower surface density of viable cells, which is less than 103 MPN·cm−2 throughout the exposure periods, indicates a high killing efficiency of the attached bacterial cells. As a result, it is easily concluded that the high concentration of quaternary ammonium compounds of quaternized PANI layers with a 12373



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Figure 8. Tafel plots of (a) pristine MS coupons after 3, 10, and 21 days of exposure to sterile SSMB medium, (b) pristine MS, (c) MS-c-PANI, and (d) MS-c-QPANI−PVBC coupons after exposure to D. desulf uricans inoculated SSMB medium for 3, 10, and 21 days.

Figure 9. Nyquist plots of (a) pristine MS coupons after 3, 10, and 21 days of exposure to sterile SSMB medium, (b) pristine MS, (c) MS-c-PANI, and (d) MS-c-QPANI−PVBC coupons after exposure to D. desulf uricans inoculated SSMB medium for 3, 10, and 21 days.

PVBC coupons, which display at least 50-fold reduction throughout in comparison with those of the pristine MS coupons (Supporting Information, Table S1).

The substantially enhanced anticorrosive properties of the MS-c-QPANI−PVBC coupons, as compared to the MS-c-PANI coupons, are not only associated with the doping of PANI 12374



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(Supporting Information). The goodness of the fitting is judged by the chi-square (χ2) values. For the pristine MS coupons in the sterile SSMB medium, the charge transfer resistance, Rct, remains quite steady in magnitude throughout the exposure period, while the resistance of the surface film, Rf, increases gradually with exposure time (Supporting Information, Table S2), attributable to the formation of the conditioning layers and iron oxides. On the other hand, both the Rct and Rf values of the pristine MS coupons decrease with exposure time during the initial 10 days in the D. desulf uricans inoculated SSMB medium, and then slightly increase with exposure time after 21 days of exposure (Supporting Information, Table S2), due to dense corrosion deposits (including biofilms and iron sulfides) formed on the MS surface. Moreover, both the values of Rct and Rf of the inoculated MS coupons are much smaller than those of the corresponding pristine MS coupons in the sterile medium, indicative of the enhancement of corrosion process by the synergistic attack of D. desulf uricans and aggressive Cl− and S2− ions. The above results confirms that the corrosion of steelbased substrates can be substantially enhanced by SRB.67 As for the MS-c-PANI and MS-c-QPANI−PVBC coupons, the much larger Rct values than those of the pristine MS coupons throughout indicates the significant reduction in the corrosion rate of the MS substrates under the protection of polymeric coatings (Supporting Information, Table S2). On the other hand, both the Rct and Rpo values of the MS-c-QPANI− PVBC coupons are larger by around 2-fold than the corresponding MS-c-PANI coupons throughout the exposure periods, indicating that the doping of PANI layer and the formation of hydrophobic PVBC layer can substantially enhance the barrier capability and the corrosion resistance of the coatings against the penetration of active anions (i.e., Cl− and S2−) and the attack by D. desulf uricans. The capacitance (Qc) of the MS-c-PANI coupons undergoes a slight increase in magnitude with exposure time (Supporting Information, Table S2), implying that the water molecules and the electrolyte ions have penetrated into the EM base PANI network upon increasing the exposure period. This leads to a notable decrease in stability and protective ability of the EM base PANI layers, as both the Rct and Rpo values of the MS-c-PANI coupons undergo a marked decrease with exposure time. However, the capacitance (Qc) of the MS-c-QPANI−PVBC coupons remains rather stable in magnitude, and even undergoes a slight decrease throughout the exposure period, indicative of the improved stability and high repelling ability to the water (and/ or electrolyte ions) penetration. This phenomenon is mainly associated with the good stability of doped PANI layers and the hydrophobicity of the outer PVBC layers. It is worthwhile to emphasize that the hydrophobic PVBC layers can not only act as a barrier to prevent undoping of the PANI layers, which can maintain the conductive state of PANI layers, upon long-time exposure to an aggressive aqueous solution, but also render a high repelling ability and corrosion resistance to the MS substrates against the synergistic attack of aggressive anions (i.e., Cl− and S2−) and SRB. 3.6. Stability and Durability of Functionalized Surfaces. Generally, organic coatings are softer than the inorganic ones; hence they can be readily damaged. As cracks and crevices in organic coatings have been widely recognized to promote localized dissolution, which can be even more serious than that produced by biofilms, it is worthwhile to emphasize the stability and durability of thermally cured polymeric

layers via the thermally induced N-alkylation reaction, but also the formation of a hydrophobic PVBC layer. Consequently, the inhibition efficiency (η) of the MS-c-PANI and MS-c-QPANI− PVBC coupons remained higher than 91 and 97%, respectively, after 21 days of exposure in the D. desulf uricans inoculated SSMB medium. On the other hand, the η values of the MS-cQPANI−PVBC coupons are always higher than those of the corresponding MS-c-PANI coupons. This further confirms that the anticorrosion capability of the EM base PANI layers in the D. desulf uricans inoculated SSMB medium is enhanced by the grafting of PBVC alkyl chains onto the quinonoid imine nitrogen of PANI. 3.5.2. Electrochemical Impedance Spectra (EIS). Electrochemical impedance spectroscopy (EIS) is a nondestructive and effective technique to determine the barrier properties of polymeric coatings.66 Figure 9 shows the respective EIS data of the pristine MS and PANI-modified MS coupons after various exposure periods in the sterile and the D. desulf uricans inoculated SSMB medium. The corresponding Bode plots are shown in Figure S4 (Supporting Information). The diameters of the semicircular loops increase with exposure time for the pristine MS coupons in the sterile SSMB medium (Figure 9a), indicative of the increase in the polarization resistance due to the formation of a conditioning layer and corrosion oxide layers on the MS coupon surface. The diameters of the semicircular loops decrease initially and then increase with exposure time for the MS coupons in D. desulf uricans (Figure 9b), implying the acceleration in corrosion under the synergistic effect of bacteria and aggressive anions (S2− and Cl−). The increase in the polarization resistance for the 21-day-exposed MS coupons is associated with the deposition of biofilm and ferrous sulfide (FexSy) on the MS surfaces. The diameters of the semicircular loops of the MS-c-PANI coupons are much larger than those of the pristine MS coupons (Figure 9c), indicating that the thermally cured PANI layers endow high corrosion resistance to the underlying MS substrates, although the polarization resistance of the MS-c-PANI coupons decreases with exposure time. The Nyquist plots of the MS-c-QPANI−PVBC coupons show two semicircles, of which the small semicircular loop at high frequency is probably attributed to the outer PVBC layer. The increase in the diameters of the semicircular loops of the MS-c-QPANI−PVBC coupons are more pronounced than those of the pristine MS and MS-c-PANI coupons, indicative of the substantial enhancement in the polarization resistance of the QPANI−PVBC bilayer coatings. The EIS data are usually fitted using proper equivalent electrical circuits (EECs) with the program EQUIVCRT of Boukamp to gain a better insight into the anticorrosive ability of the PANI layer and QPANI−PVBC bilayer coatings. Two types of EECs were utilized to fit the EIS data of the pristine MS and PANI-modified MS coupons, respectively, as shown in Figure S5 (Supporting Information). For the circuit elements, the pore resistance, Rpo, represents the extent of ionic conduction through a polymeric coating in an electrolyte environment. It is widely used as a criterion to evaluate the protection capability and efficiency derived from polymeric coatings, while the constant phase element (CPE) of coating, Qc, is used instead of the coating capacitance (Cc) due to surface heterogeneity and diffusion process. The capacitance or CPE of polymeric coatings provides information on the extent of water uptake and the stability of coatings.66 The fitted parameters of the EIS data are summarized in Table S2 12375



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Notes

coatings. For the MS-c-QPANI−PVBC coupons after exposure in the D. desulf uricans inoculated medium for 21 days, the XPS results reveal that the composition of the functionalized surface remains almost unchanged (Supporting Information, Figure S6), indicating that the thermally cured QPANI−PVBC bilayers on the MS substrates are relatively stable and durable. Moreover, silanization of metallic substrates for passivation is a well-established process. The silane monolayer affixed to the substrates via robust Si−O bonds has been widely recognized to be stable in electrolyte solutions.68 Additionally, the PANI layers were deposited on the silanized MS via thermal curing reaction between the epoxy groups of GPTS and the imine groups of the PANI. The thermally cured polymers have also been verified to be stable on various substrates under harsh environments.55,56 Further studies are in progress to focus on the long-term performance of the QPANI−PVBC bilayer coatings tethered on the MS substrates for the mitigation of the SRB-induced corrosion in open seawater field environments.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the financial assistance of a key project of the National Natural Science Foundation of China (No. 21236004).

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(1) Duraibabu, D.; Ganeshbabu, T.; Manjumeena, R.; Ananda Kumar, S.; Dasan, P. Unique coating formulation for corrosion and microbial prevention of mild steel. Prog. Org. Coat. 2014, 77, 657−664. (2) Dall’agnol, L. T.; Cordas, C. M.; Moura, J. J. Influence of respiratory substrate in carbon steel corrosion by a Sulfate Reducing Prokaryote model organism. Bioelectrochemistry 2014, 97, 43−51. (3) Schutz, M. K.; Moreira, R.; Bildstein, O.; Lartigue, J. E.; Schlegel, M. L.; Tribollet, B.; Vivier, V.; Libert, M. Combined geochemical and electrochemical methodology to quantify corrosion of carbon steel by bacterial activity. Bioelectrochemistry 2014, 97, 61−68. (4) Stipaničev, M.; Turcu, F.; Esnault, L.; Schweitzer, E. W.; Kilian, R.; Basseguy, R. Corrosion behavior of carbon steel in presence of sulfate-reducing bacteria in seawater environment. Electrochim. Acta 2013, 113, 390−406. (5) Noor, N. M.; Yahaya, N.; Abdullah, A.; Tahir, M. M.; Sing, L. K. Microbiologically influenced corrosion of X-70 carbon steel by Desulfovibrio Vulgaris. Adv. Sci. Lett. 2012, 13, 312−316. (6) Alasvand, K. Z.; Rai, V. R. Microorganisms: induction and inhibition of corrosion in metals. Int. Biodeterior. Biodegrad. 2014, 87, 66−74. (7) Kakooei, S.; Ismail, M. C.; Ariwahjoedi, B. Mechanisms of microbiologically influenced corrosion: a review. World Appl. Sci. J. 2012, 17, 524−531. (8) Ashassi-Sorkhabi, H.; Moradi-Haghighi, M.; Zarrini, G.; Javaherdashti, R. Corrosion behavior of carbon steel in the presence of two novel iron-oxidizing bacteria isolated from sewage treatment plants. Biodegradation 2012, 23, 69−79. (9) AlAbbas, F. M.; Williamson, C.; Bhola, S. M.; Spear, J. R.; Olson, D. L.; Mishra, B.; Kakpovbia, A. E. Influence of sulfate reducing bacterial biofilm on corrosion behavior of low-alloy, high-strength steel (API-5L X80). Int. Biodeterior. Biodegrad. 2013, 78, 34−42. (10) Lewandowski, Z.; Beyenal, H. Mechanisms of microbially influenced corrosion. In Marine and Industrial Biofouling; Springer: Berlin, 2009; pp 35−64. (11) Little, B. J.; Mansfeld, F. B.; Arps, P. J.; Earthman, J. C. Microbiologically Influenced Corrosion; Wiley: Hoboken, NJ, 2007; pp 72−83. (12) Lata, S.; Sharma, C.; Singh, A. K. Microbial influenced corrosion by thermophilic bacteria. Cent. Eur. J. Eng. 2011, 2, 113−122. (13) Hamilton, W. Sulfate-reducing bacteria and anaerobic corrosion. Annu. Rev. Microbiol. 1985, 39, 195−217. (14) Fathy, M.; Badawi, A.; Mazrouaa, A. M.; Mansour, N. A.; Ghazy, E. A.; Elsabee, M. Z. Styrene N-vinylpyrrolidone metal-nanocomposites as antibacterial coatings against sulfate reducing bacteria. Mater. Sci. Eng., C 2013, 33, 4063−4070. (15) Beech, I. B. Sulfate-reducing bacteria in biofilms on metallic materials and corrosion. Microbiol. Today 2003, 30, 115−117. (16) Iverson, W. P. Direct evidence for the cathodic depolarization theory of bacterial corrosion. Science 1966, 151, 986−988. (17) King, R. A.; Miller, J. D. A. Corrosion by the sulfate-reducing bacteria. Nature 1971, 233, 491−492. (18) King, R.; Miller, J.; Smith, J. Corrosion of mild steel by iron sulfides. Br. Corros. J. 1973, 8, 137−141. (19) Iverson, W. P. Corrosion of iron and formation of iron phosphide by Desulfovibrio desulf uricans. Nature 1968, 217, 1265− 1267. (20) Beech, I. B.; Sunner, J. Biocorrosion: towards understanding interactions between biofilms and metals. Curr. Opin. Biotechnol. 2004, 15, 181−186.

4. CONCLUSIONS A facile and environmentally benign approach based on thermally induced reaction was developed to synthesize antibacterial and hydrophobic QPANI−PVBC bilayer coatings on mild steel (MS) substrates for mitigating SRB-induced corrosion in artificial seawater. The functionalization process involved (i) covalent immobilization of a silane monolayer containing the terminal epoxy groups as anchor sites, (ii) thermally induced curing of PANI onto the MS substrates by a coupling reaction between the epoxy groups and amine groups (−NH−) of PANI, and (iii) thermally induced N-alkylation of quinonoid imine of PANI by vinylbenzyl chloride (VBzCl) to produce biocidal functionality, accompanied by the formation of a hydrophobic PVBC layer. The so-synthesized bilayer coatings exhibited a desired high efficiency in inhibiting bacterial adhesion and proliferation, as revealed by the bacterial adhesion and viability assays. The electrochemical results revealed that the QPANI−PVBC bilayer coatings also rendered high barrier ability and corrosion resistance to the underlying MS substrates against the synergistic attack of aggressive anions (i.e., Cl− and S2−) and D. desulf uricans in harsh simulated marine environments.

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ASSOCIATED CONTENT

S Supporting Information *

Procedures of MS coupon preparation, synthesis of the emeraldine base PANI, and SRB cultivation and inoculation are described in detail in the experimental section. The experimental setup for the MIC studies is shown in Figure S1. Electrochemical results: analysis parameters of Tafel plots (Table S1), fitted parameters of EIS spectra (Table S2), and Bode plots of pristine MS and surface-functionalized MS coupons (Figure S4) and corresponding equivalent electric circuits for EIS spectra fitting (Figure S5). Surface characterization: XPS spectra of hydroxylated (MS-OH) surface (Figure S2) and MS-c-QPANI−PVBC surface after 21 days of exposure (Figure S6); representative FM images for pristine MS coupons with bacterial attachment (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-28-85999978. Fax: +86-28-85460556. E-mail: [email protected]. 12376



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Surface Modification of Mild Steel with Thermally Cured Antibacterial

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