A Two-Step Silane Coating Incorporated with Quaternary Ammonium Silane for Mitigation of Microbial Corrosion of Mild Steel.

Quaternary ammonium compounds have been used as antibacterial materials. However, as they are hydrophilic and produce a positively charged surface, it is challenging to develop a durable antimicrobial coating of such compounds. The objective of this study is to investigate a two-step silane coating incorporated with quaternary ammonium silane for mitigation of microbiologically influenced corrosion (MIC) of mild steel in biotic solution (a marine environment with bacteria). The corrosion resistance was characterized by electrochemical impedance spectroscopy and potentiodynamic polarization tests. The intact silane coating and that pre-exposed to the biotic solution were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The most probable method (MPN) was used to quantify the active microorganisms attached to the uncoated and silane-coated surfaces. Electrochemical results reveal that the coating thus developed improved the corrosion resistance of steel in the biotic solution. The MPN, FTIR, and scanning electron microscopy suggest a significant decrease in the number of active cells that get attached to the coated surface.

Introduction

Biocorrosion or microbiologically influenced corrosion (MIC) is the deterioration of metals and alloys caused or accelerated by the existence of bacteria and other microbes and their activities.[1] During MIC, synergistic interactions often take place between the surface of the metal, corrosion products, and bacterial cells and their metabolites.[2] The most important features of MIC are related to bacterial activity through producing corrosive metabolites, for instance, the formation of H2S by sulfate-reducing bacteria (SRB).[3] SRB are among the microorganisms that are responsible for MIC of metals and alloys including cast iron, carbon steel, low alloy steels, stainless steels, high nickel alloys, and copper alloys.[4]

When industrial equipment is in service for a long time, MIC becomes a common risk factor for an accident (e.g., fluid flooding, leakage, rupture, etc.).[5] The total direct annual cost because of corrosion in the United States was estimated to be $276 billion, which is 3.1% of the world’s gross domestic product (GDP).[6] To the oil and gas industry alone, the estimated annual cost of corrosion was about $13.4 billion in 2001, of which about $2 billion was due to MIC.[7] Escape of more than 100,000 tons of methane from a well casing in a storage field in Aliso Canyon, CA, USA and leakage of 750,000 L of oil from the oil pipeline in Prudhoe Bay, Alaska are among the examples of disastrous MIC failure caused by methanogens and SRB, respectively.[8] Many strategies have been practiced to mitigate MIC such as biocide treatments, development of beneficial biofilms using bacteria that produce antibacterial properties and corrosion inhibitors, forming of protective passive oxides within biofilm matrices,[9] coatings using silicone compounds, epoxy resins, and fluorinated compounds, choosing alloys with necessary resistance to MIC-assisted pitting and crevices, and cathodic protection and monitoring of MIC.[10]

Sol–gel coatings can be used for corrosion protection and combined with other environment-friendly and non-hazardous materials for mitigation of MIC. Gottenbos et al.[11] investigated microbial activities of a covalently coupled quaternary ammonium silane (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) coating on silicon rubber. The antibacterial activity of the silicone rubber with and without coatings was tested in the presence of different types of bacteria (Staphylococcus epidermidis HBH2 102, Gram-positive Staphylococcus aureus (S. aureus) ATCC 12600, Pseudomonas aeruginosa AK1 12600, and Gram-negative Escherichia coli (E. coli) O2K2). The coating showed antimicrobial activity toward all the tested bacterial strains in vitro, which was confirmed in vivo for the S. aureus strain. They found that staphylococcal killing was significant, while S. aureus were not so effectively killed. Yuan et al.[12] prepared antibacterial inorganic–organic hybrid coatings, consisting of a cross-linked polysilsesquioxane inner layer and quaternarized poly(2-(dimethyamino)ethyl methacrylate) outer blocks. The coating thus formed on stainless steel was found to be durable and resistant to corrosive species due to the inner layer of polysilsesquioxane. The outer quaternarized layer provided antibacterial characteristics to the steel surface to inhibit MIC in the presence of SRB in a seawater medium. Marini et al.[13] prepared antibacterial hybrid coatings containing a quaternary ammonium salt that is covalently bonded to the organic–inorganic network. They applied a sol–gel coating on plastics to test the antibacterial activity of coated samples that were exposed to both Gram-negative (Escherichia coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 6538) media. After 24 h of exposure, the viable count for E. coli decreased dramatically by 61.3%, and after 48 h, almost the entire bacteria disappeared. Similarly, a remarkable decrease (97.9%) in S. aureus strains was observed in the case of quaternary ammonium salt-coated samples in 24 h tests. Less than 1% of S. aureus were detected after 48 h. Recently, Wang et al.[14] synthesized a novel diblock copolymer incorporating antibacterial quaternary ammonium groups as pendant groups, poly(3-(methacryloylamino) propyltrimethyl ammonium chloride)-b-poly(styrene) (PMS), using interfacial polymerization. They reported that via modifying the ratios of monomers and toluene/styrene, PMS anisotropic particles (APs) could be successfully obtained based on different assembly behaviors. To improve the antibacterial activity of composite materials, silver-loaded chitosan (Ag@CS) and PMS APs were combined to prepare natural/synthetic polymer antibacterial materials with dual-active centers (Ag@CS/PMS-4 APs).

A common characteristic of antibacterial compounds, such as quaternary ammonium compounds, is their ability to bind to the cell membrane, disrupt its function to become semi-permeable, and cause cell leakage.[15] It is reported that[16,17] quaternary ammonium compounds (such as quaternary ammonium resins) interact electrostatically with the negatively charged surfaces of bacteria followed by permeation of the alkyl chain into the bacterial membranes leading to bacterial death due to the dissolution of the bacterial cells. Though the quaternary ammonium compounds are reported[11,18−23] to possess an antimicrobial property, the protonation of amino groups enhances the migration of chloride ions toward the metal surface; thereby, the durability of such a coating in a corrosive environment could be compromised.[24−27] There is little reported on the development of a silane coating containing quaternary ammonium silane to improve the corrosion resistance of mild steel in the marine environment with bacteria. Al-Saadi and Raman[26] investigated the effect of the coatings developed upon two-step silane treatment of the quaternary ammonium silane on corrosion resistance of mild steel in an aerobic 0.6 M NaCl solution without bacteria. In the present study, the inhibition action of the two-step silane coating of a non-functional silane, bis[triethoxysilyl]ethane (BTSE), followed by another top coating with a silane mixture of bis-[trimethoxysilylpropyl]amine (bis-amino silane) and 3-[trimethoxysilyl]-propyldimethyloctadecyl ammonium chloride (QAS) in a mixing ratio of 5:1 (v/v) was evaluated for resistance of mild steel to MIC in a chloride solution with sulfate-reducing bacteria (SRB). Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization measurements (PDP) were employed for assessment of corrosion behavior of uncoated and two-step silane-coated mild steel. To quantify the active SRB that are attached to the untreated and silane-treated mild steel during the duration of exposure to the marine environment inoculated with bacteria (i.e., biotic solution), the most probable number (MPN) method was used. After pre-exposure to the biotic solution, the mild steel specimens with and without a silane coating were characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM).

Experimental Methods

Preparation of Silane Solutions

Neat silanes of BTSE, bis-amino silane, and quaternary ammonium silane (QAS) were purchased from Gelest, Inc. To prepare the stable hydrolyzed BTSE solution, 4% BTSE, 0.4% acetic acid, 6% deionized water, and 89.6% methanol by volume were mixed in that order as described by Subramanian and van Ooij.[27] The BTSE solution was stirred for 1 h and held for 48 h without stirring before use.

A 4% bis-amino silane solution was prepared by mixing 4% (vol %) silane with an ethanol and deionized water mixture. The ratio of deionized water to ethanol was 6/90 (v/v). The pH of the solution was lowered to be 7.5 using acetic acid, and the solution was held still for 24 h.

In the case of QAS, the neat silane was added drop-by-drop to a mixture of ethanol and deionized water. The ratio of silane/deionized water/ethanol was 4/6/90 (v/v/v). Hydrolysis of QAS was carried out at normal pH (i.e., pH = 6.3), and the solution was aged for 24 h before use.

Steel Sample Preparation

A copper wire was soldered to the mild steel coupon (25 mm × 25 mm × 12 mm), and a glass tube was used to encase the wire. The coupons were mounted in a non-conducting epoxy resin. Before the silane coating, the coupons were ground with a 2500 grit SiC paper, degreased with acetone and ethanol, and dried with compressed air. The cleaned coupons were immersed in a sodium hydroxide solution (NaOH, 2.5% (w/w)) for 10 min to improve the wettability of the coupon surface. Then, they were rinsed with deionized water and dried with compressed air.

For silane coating, the alkali-pre-treated coupons were dipped in the BTSE silane solution for 25 min followed by another dipping for 5 min in the silane mixture of bis-amino silane and QAS. The second silane mixture was prepared by mixing the bis-amino silane and QAS in a ratio of 5/1 (v/v). The mixture was stirred for 10 min at room temperature before use. The two-step silane-coated coupons were cured for 1 h at 120 °C. To avoid crevice corrosion during electrochemical tests, the interfaces between the metal and non-conducting resin were coated with an enamel coating and allowed to dry for 12 h in a desiccator. The area left to be exposed to the electrolyte was 1 cm2. Nomenclature for the two-step silane coating is B(BQ,npH)5:1. The first B is for BTSE silane, the second B for bis-amino silane, Q for QAS, npH denotes a normal pH (QAS is hydrolyzed at normal pH), and 5:1 is the volume ratio of the bis-amino silane to the QAS.

Design of the Anaerobic Electrochemical Cell

Wide-neck laboratory bottles (DURAN GLS 80) were used to fabricate the three-electrode glass electrochemical cells with a volume of 250 mL (Figure ). Uncoated and silane-coated mild steel specimens were used as working electrodes. A saturated calomel electrode (SCE) that was attached to a salt bridge and a platinum mesh were used as reference and counter electrodes, respectively. A modified Postgate C medium inoculated with SRB was used to simulate the marine environment with bacteria (biotic solution) in which all the ingredients, as shown in Table , were mixed and the pH of the solution was maintained to be 7.5 by adding 3 M NaOH solution. The modified Postgate C solution was autoclaved for 20 min and then allowed to cool down to room temperature before use.

Figure 1

Schematic diagram of the anaerobic three-electrode electrochemical cell.

Table 1

The Composition of the Modified Formula of Postgate C Solution

ingredients	composition (g/L)
potassium dihydrogen phosphate (KH2PO4)	0.5
ammonium chloride (NH4Cl)	1
sodium sulfate (Na2SO4)	4.5
calcium chloride (CaCl2·6H2O)	0.06
magnesium sulfate heptahydrate (MgSO4·7H2O)	0.06
sodium citrate	0.5
yeast extract	1
sodium chloride (NaCl)	35
ferrous sulfate (FeSO4)	0.004
lactic acid (88%)	4 mL
water	1000 mL

Before electrochemical tests, a 1 mL aliquot of the three-day-old SRB, which were pre-cultured in the modified Postgate B solution,[28] was inoculated under a N2 atmosphere in a 200 mL solution of the modified Postgate C in the sterilized electrochemical cells and incubated at 30 °C for 3 days. A thick layer of sterilized paraffin oil was placed on the top of the liquid medium after the inoculation of SRB culture. The most probable number (MPN) method was used to quantify the survival of bacteria, and it was determined to be >105 MPN/L. Before assembling the electrochemical cell, the silane-coated specimen was immersed in 70% ethanol for 3 h for sterilization followed by drying with N2. The uncoated specimens were sterilized in absolute ethanol for 5 min in an ultrasonic bath followed by immersion in 70% ethanol for 15 min to avoid iron oxide formation. The platinum electrode and a luggin tube were autoclaved for 20 min before fitting them into the cell. The luggin tube was filled with sterilized agar and KCl to be used as a salt bridge, and the saturated calomel electrode was immersed in the luggin tube. A semi-continuous mode was employed for bacterial growth, where 75% of the modified Postgate C solution was replaced with an equal amount of the fresh and sterilized medium every four days[29] followed by purging of the electrolyte with nitrogen for 15 min.

Electrochemical Measurements

The three-electrode glass electrochemical cell, which is described in Section , was connected to a Princeton Applied Research potentiostat (model 2273) to perform the electrochemical measurements (i.e., PDP and EIS). The EIS measurements were carried out on the specimens at different durations of exposure to the modified Postgate C solution with and without SRB, by applying a small-amplitude sinusoidal potential of 10 mV at an open-circuit potential. The impedances were measured in the frequency range of 1 MHz to 10 mHz where 10 points per decade of frequency were recorded. Applying a small-amplitude sinusoidal potential of 5–10 mV does not damage the biofilm developed on the metal surface or affect the growth of bacteria.[30−33] For the PDP measurements, the scan was started at a potential of 250 mV below the open-circuit potential using a scan rate of 0.166 mV/s. To examine the reproducibility, measurements were repeated at least three times for each of the experimental conditions.

Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR)

ATR-FTIR spectra were collected using a PerkinElmer Spectrum 100 series FTIR spectrometer in the mid-IR range from 4000 to 400 cm–1. The spectra were collected for the intact silane deposited on the steel surface, the residue of the biotic medium, and the corrosion product film that developed on the silane-coated specimen pre-exposed to the anaerobic chloride solution with SRB (biotic). For each test, 64 scans were performed with a spectral resolution of 4 cm–1. Prior to the collection of FTIR spectra, background scans were performed to be subtracted from the collected FTIR spectra.

Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy (EDS)

Morphologies of the uncoated and two-step silane-coated specimens were examined by scanning electron microscopy (SEM) at 15 keV with an EDS analyzer. To avoid charging, the specimens pre-exposed to a corrosive environment were gold-coated prior to the SEM test.

Results and Discussion

Coating Morphology, Thickness, and EDS Analysis

The SEM micrographs and EDS spectra of the silane film formed on the mild steel surface upon the two-step treatments (B(BQ,npH)5:1) are shown in Figure a–d. The mild steel surface appears to be entirely covered with a transparent, shiny, and crack-free silane film; however, tiny dark clots were observed (Figure a). The scratches on the underlying steel surfaces are caused by the grinding. Though EDS analysis (Figure c,d) exhibited high Si peaks at two different locations of the silane film on the mild steel surface (S1 and S2), the intensity of Si peaks was stronger for the tiny dark clots.

Figure 2

(a,b) SEM micrographs of the two-step silane-coated mild steel prior to exposure to biotic solution and coating thickness. (c,d) EDX spectra of the two-step silane coating.

To find the thickness of the silane film developed on the steel surface, the cross section of a coated sample was cold mounted and ground with a 2500 grit SiC paper followed by fine polishing with a diamond suspension up to 1 μm. The sample was subsequently washed with deionized water and ethanol and dried with compressed air. The specimen was gold-coated prior to SEM to avoid charging. The B(BQ,npH)5:1 coating deposited on the mild steel surface (Figure b) was relatively uniform with an average thickness of 15 μm.

Electrochemical Measurement

The impedance spectra (i.e., the Nyquist and Bode phase plots) for the uncoated mild steel specimens pre-exposed to the biotic solution (the modified Postgate C medium inoculated with SRB) for 3, 7, and 14 days are shown in Figure a,b. As references, the spectra for the uncoated specimen pre-immersed in the abiotic solution for 14 days are shown in Figure a,b, as well. The impedances (i.e., a broad measure of corrosion resistance) of the uncoated specimens that were pre-exposed to biotic solution for 3 days and abiotic solution for 14 days are similar, suggesting an insignificant damaging influence of bacteria for 3 days. However, corrosion resistance of the uncoated steel in biotic solution decreased with a further increase in duration of exposure, i.e., from 17.8 kΩ cm2 at 3 days to 8 kΩ cm2 at 7 days, and after 14 days, the impedance dropped down to be 4 kΩ cm2. A single and broad time constant is observed in the frequency range of 100 to 103 for the uncoated specimen pre-exposed to the biotic medium for 3 days (Figure b), which corresponds to the development of an aqueous corrosion product of steel such as iron oxide/hydroxide and initiation of a biofilm. The second time constant begins to emerge in the low-frequency regime when the duration of exposure increased to 7 days. The second time constant becomes quite prominent upon increasing the exposure duration to 14 days. The emergence of the second time constant in the low-frequency regime in the biotic medium and its increasing prominence with time indicate the development of additional corrosion activity at the metal/electrolyte interface,[28,33−38] presumably, as a result of biological activity. This activity can be attributed to the reaction of the steel substrate with the biogenic sulfide.[28,34] The high-frequency time constant corresponds to the development of a common corrosion product of the aqueous corrosion of steel such as iron oxide/hydroxide and the biofilm.[28,34,39,40]

Figure 3

EIS spectra of uncoated and silane-coated mild steel after exposure times of 3, 7, and 14 days in a marine environment inoculated with SRB (biotic solution). (a,c) Nyquist plots and (b,d) Bode phase plots (the spectrum of uncoated mild steel pre-immersed in abiotic solution for 14 days is shown for comparison). (a) and (b) are adapted from Al-Saadi et al.[28]

EIS results for the two-step silane-coated mild steel (B(BQ,npH)5:1) that was pre-exposed to the aqueous chloride environment inoculated with SRB for 3, 7, and 14 days are presented in Figure c,d. As evident from Figure a,c, impedances |Z| of the silane-treated samples were considerably higher than those for uncoated specimens, clearly establishing the two-step silane treatment to have considerably improved the corrosion resistance of steel in the biotic medium. For instance, the corrosion resistance of the silane-coated sample during the first 3 days of pre-exposure to SRB media (i.e., 69.4 kΩ cm2) was nearly four times higher than that of the uncoated steel (17.8 kΩ cm2). Though the corrosion resistance of the two-step coated steel decreases with increasing duration of pre-exposure to the biotic solution, the coated steel still shows considerable resistance even at the end of the period of pre-exposure (i.e., 14 days). For exposure for 14 days in the biotic medium, the impedance of the two-step coated steel was about five times higher than that of the uncoated steel. This improvement is attributed to the covalent bonding of BTSE silane to the alkali-pre-treated steel surface. The six silanol groups of the fully hydrolyzed BTSE (at pH 4) react with the basic hydroxyl groups of the alkali-pre-treated steel surface to form stronger covalent bonds that are less susceptible to hydrolysis.[41] Another advantage of using the two-step route is the relatively wider range of pH that is applicable in the second step of silane treatment (i.e., with QAS), i.e., unlike the first step where pH needed to be in the range where metal hydroxide was stable to enable bonding with the hydrolyzed silane film.[42] Regarding the mitigation of bacterial attachment, the positively charged surface that formed due to the QAS film is known to interact with the cell membrane of bacteria causing cell membrane disruption and cell death.[19,20,43]

Similar to the uncoated steel, a single time constant is observed for the two-step coated steel pre-exposed to the biotic medium for 3 days (Figure d); however, this is less broad (cf. that for the uncoated steel), presumably, because this corresponds to the existing two-step coating (cf. development of corrosion in the case of the uncoated steel). The second time constant begins to emerge when the time exposure increased to 7 days; however, the second time constant for the two-step coated steel emerges in the high-frequency regime (as opposed to that in the low-frequency regime for uncoated steel in Figure b). The development of the second time constant in the high-frequency regime can only be attributed to development of a biofilm at the coating surface and its increasing prominence when exposure time increased to 14 days. The less prominence of the second time constant indicates effectiveness of QAS causing formation of a less robust biofilm, which is confirmed by the bacterial count data (described subsequently).

It is emphasized that the Bode phase plots in Figure b,d provide evidence for the distinct mechanistic differences in the uncoated steel and two-step coated steel systems. The two time constants for uncoated steel correspond to the aqueous corrosion products of steel and the biofilm (i.e., the medium-frequency time constant) and the reaction of the biogenic sulfide of the steel substrate (i.e., the low-frequency regime). On the other hand, the two time constants for the two-step coated steel correspond to robust coating (i.e., the medium-frequency time constant) and a less robust biofilm (i.e., the high-frequency regime). It is important to note that the frequency regimes for the medium-frequency time constants for the uncoated and coated steel are distinctly different (Figure b,d), which is consistent with the description that in the case of coated steel, this time constant corresponds to the two-step coating, whereas this time constant corresponds to the aqueous corrosion products of steel and the biofilm for uncoated steel.

As described in Figure S1a,b (in the Supporting Information document), two equivalent electrical circuit models (EECs) are required for explaining the mechanisms operating at the different interfaces (i.e., corrosion product(s) induced by the biofilm/electrolyte and the metal/electrolyte) for the uncoated steel at different durations of pre-exposure to the biotic medium. A classic Randles circuit (Figure S1a) is used to explain the electrode kinetics of the uncoated steel pre-immersed for 3 days in the chloride environment with bacteria. However, an additional time constant (to account for the resistance of corrosion products and the constant-phase element of corrosion products) and the Warburg element (Figure S1b) were required in the EEC model to explain the electrode kinetics of the uncoated steel pre-exposed for 7 and 14 days. The Warburg element (W) accounts for the diffusion processes through the defects in the bacterial biofilm developed on the metal surface. For the two-step silane treatments (B(BQ,npH)5:1), the EIS data were found to fit the electrical equivalent circuit shown in Figure S1c. According to this model, at relatively high frequencies, the coating properties and the changes that take place during exposure to the corrosive media can be characterized, whereas at relatively low frequencies, the corrosion reaction at the metal surface can be assessed.[44] Thus, the proposed model for silane-treated steel consists of the solution resistance (RS), connected in series with the parallel connection of the constant-phase element of the silane film (QC), the coating resistance (RC), which is connected with the parallel connection of the double-layer constant-phase element (Qdl), and the charge transfer resistance (Rct). The examples of the extent and quality of the fits and the agreements between the experimental and the simulated data for uncoated and the two-step silane-coated steel pre-exposed to the chloride environment inoculated with SRB for 7 days are presented in Figure . The agreement between the experimental and the simulated data for other EIS measurements (i.e., 3 and 14 days) is presented in Figure S2 in the Supporting Information document.

Figure 4

Examples of the agreement between the experimental and calculated data of EIS measurements of (a) uncoated and (b) silane-coated mild steel pre-exposed to biotic media for 7 days.

The evolutions of the simulated parameters of the EECs (in Figure S1a,b) are depicted in Figure . The charge transfer resistance (Rct) of the uncoated steel, in the chloride environment with bacteria, decreased considerably (i.e., 15 times) as the duration of exposure increased from 3 to 14 days (Figure a), whereas the Qdl of uncoated steel increased with the exposure duration (Figure b). The simultaneous decrease in Rct and increase in Qdl in the case of steels exposed to biotic marine solutions are due to the deposition of a conductive ferrous sulfide film on the metal surface.[45,46] Because the biofilm and corrosion products may not provide complete coverage on the steel surface, both Rf and Qf increased during the pre-exposure periods for 7 and 14 days, as shown in Figure a,b. For two-step silane coatings that are incorporated with the quaternary ammonium silane (B(BQ,npH)5:1), the time-dependent evolutions of the different interfacial parameters associated with the proposed EEC are shown in Figure c,d. The slower decrease in coating resistance (RC) and the sluggish increase in coating capacitance (QC) suggest a slow uptake of the electrolyte and its slow migration through the pores of the coating during immersion in the chloride solution inoculated with bacteria.[34,47] Furthermore, the insignificant decrease in Rct and Qdl during the period of pre-immersion up to 7 days infers the retardation in degradation due to the coating developed by two-step treatment. While Rct decreases when the coated steel is exposed for 14 days to the biotic solution, the Qdl increases, which could be a direct consequence of the time-dependent delamination of the silane coating at the coating/metal interface.

Figure 5

Evolution of equivalent electrical circuit parameters obtained by fitting the EIS measurements of (a,b) uncoated and (c,d) two-step silane-coated mild steel pre-exposed to marine solution with SRB for 3, 7, and 14 days.

Figure shows the PDP plots of the uncoated and two-step silane-coated specimens pre-exposed to the chloride environment with SRB for 7 and 14 days. The plots for uncoated mild steel are adapted from ref (28) where the mechanism of microbial corrosion of uncoated steel in chloride solution inoculated with SRB is explained in detail. As the exposure time to the biotic solution increased from 7 to 14 days, the Ecorr for the uncoated steel shifted significantly toward the cathodic region (i.e., ∼128 mV more negative) compared to the little shift in that of the coated steel (∼51 mV toward the cathodic potential). Since the reduction in Ecorr is an indication of surface thermodynamic instability and the increase in susceptibility to anodic dissolution,[48] the polarization data reveal that the two-step silane-coated steel is less vulnerable to microbial corrosion and more stable than the uncoated steel in the biotic environment. The corrosion current density of the uncoated steel (4.375 μA/cm2) is in excess of an order of magnitude higher than that of the two-step silane-coated steel (0.356 μA/cm2) after immersion in the chloride-biotic solution for 14 days.

Figure 6

Polarization curves of uncoated and silane-coated mild steel after exposure times of 7 and 14 days in biotic solution. Plots for the uncoated alloy are adapted from Al-Saadi et al.[28]

Bacterial Cell Number

The most probable number (MPN) method was used to quantify the active microorganisms that get attached to the untreated and silane-treated mild steel pre-exposed to biotic media for 3, 7, and 14 days. In MPN determination, a serial dilution is used to measure the concentration of a target microbe in a sample.[49,50] After the specified period of pre-exposure to a biotic medium, the uncoated and silane-treated samples were carefully transferred from the medium into the 50 mL Duran bottle containing 10 mL of sterile phosphate buffer solution. Then, the Duran bottle was subjected to ultrasonication (frequency of 50 kHz for two and a half minutes). To get estimates over a wide range of possible concentrations, the serial dilution approach is used.[49,50]

In the present work, after the sonication, 1 mL of the solution containing the bacteria, biofilm, and other suspensions detached from the mild steel surface in phosphate buffer solution was subjected to the decimal consecutive dilutions in triplicate sterilized test tubes for nine times. These test tubes were pre-filled with 9 mL of modified Postage B prior to the inoculation of 1 mL of SRB. The modified Postage B preparation method is described in detail in the literature.[28] The inoculation procedure was carried out under a nitrogen atmosphere. Then, a film of sterilized paraffin oil was placed on top of the solution. The test tubes were kept in an incubator at 30 °C for 4 days, and the bacterial growth was monitored during the period of incubation. MPN/cm2 results were calculated using the MPN table for three tubes and taking into account the multiplication with the dilution factor as described in the literature.[49,50]

Figure illustrates the SRB population attached to uncoated and silane-coated mild steel specimens during the pre-exposure periods of 3, 7, and 14 days to biotic media. For uncoated mild steel pre-exposed for 3 days to biotic media, the number of the active cells on the surface was about 6 × 106 MPN/cm2. The number of active cells increased by more than 16 times after 7 days of pre-exposure and remained higher by seven times after 14 days. However, for silane-coated steel samples, the number of active cells adhering to the coated surfaces was much lower in comparison to those attached to the uncoated steel substrates. The lowest number of active cells of bacteria (5.5 × 104 MPN/cm2) was found to be attached to the B(BQ,npH)5:1-coated surfaces after 3 days of pre-exposure to biotic media. This can be attributed to the presence of antibacterial QAS within the coatings. The decrease in the inhibitive action of B(BQ,npH)5:1 treatment as indicated by the increase in the number of active cells through a further period of pre-exposure (7 to 14 days) is presumably due to the interaction of the hydrophilic ammonium group in QAS with water and as a result compromises the antibacterial activity and corrosion resistance of the coating with respect to the time of immersion in corrosive biotic solution.

Figure 7

Sulfate-reducing bacteria population on uncoated and two-step silane-coated mild steel surfaces with respect to the time of exposure to the biotic media.

ATR-FTIR Characterization

Figure shows the ATR-FTIR spectra of the intact two-step silane film incorporated with QAS (i.e., B(BQ,npH)5:1) that was deposited on mild steel, the residues collected from the 7 days-old biotic media, and the film developed on the silane-coated mild steel exposed to the biotic solution for 7 days. For the intact silane film, the broad band between 3050 and 3250 cm–1 is due to the asymmetric and symmetric NH2 stretching.[51] The other amino group peaks noticed at 1572, 756, and 692 cm–1 are attributed to the distorted NH2 group,[27] free NH, and protonated −NH2+, respectively.[52] Three peaks correspond to the C–H symmetric and asymmetric stretching due to −CH2– modes: two peaks at 2848 and 1460 cm–1 are assigned to the C–H symmetric stretching[53,54] and one for C–H asymmetric stretching at 2918 cm–1.[53] In the spectrum, the most pronounced peak noticed at 1012 cm–1 (with a shoulder at 1072 cm–1) is due to the asymmetric Si–O–Si band,[55,56] which is indicative of the formation of a siloxane network as a result of condensation of silanols. As reported in the literature,[57] the siloxane chains due to the bis-amino system could be more linear than the structure formed using the other silane system, which is not cross-linked homogeneously (such as BTSE).[58]

Figure 8

FTIR spectra of the intact silane film deposited on the mild steel surface, the SRB bulk medium residue after 7 days, and the silane-coated mild steel substrate after pre-exposure to biotic media for 7 days.

The very dwarf peaks at 1340 and 1400 cm–1 correspond to the carboxylate ions (COO−) in the bicarbonate salt and ammonium carbamate, which are formed due to the affinity of aminated silane and acetic acid.[59] The band assignments that corresponded to amino, methylene, and siloxane groups indicate the successful formation of a two-step coating using BTSE silane (4 vol %, pH 4) followed by dipping in a mixture of bis-amino (4 vol %, pH 7.5) and QAS (4 vol %, natural pH 6.3) silanes in a mixing ratio of 5:1 (i.e., B(BQ,npH)5:1).

For the 7 days-old biotic residue and the silane-coated sample pre-immersed for 7 days in biotic solution, the details of the preparation steps prior to ATR-FTIR tests are described in the previous work of the authors’ group.[28,34]Table presents the detected characteristic peaks for the biotic residue and their assigned functional groups. The detected peaks are in three main frequency regions for fatty acids, proteins, and polysaccharides.[28,34] Two strong bands at 2920 and 2850 cm–1 of the asymmetric and symmetric stretches of CH2 are detected in the frequency regime of the functional groups assigned to fatty acids.[60−66] At the frequency regime of proteins, the bands of C=O of amide I, N–H and C–N of amide II, and C–H bend of CH2 at the frequencies of ∼1640, 1544, and 1454 cm–1 are observed, respectively.[60,61,64−66] It is reported that the detection of amide functional groups in such samples confirms the presence of the bacterial cells.[63] Also, the detection of the functional groups assigned for the extracellular polymeric substances (EPS) is an indication of the presence of living bacteria in the biotic solution. The band detected between the frequencies of ∼1160 and ∼900 cm–1 confirms the presence of EPS.[60,63] This band is detected at the frequencies of the polysaccharides and the mixed region of the proteins and polysaccharides. The other bands in the frequency range of 1400–1370 cm–1 in the mixed region are the polysaccharide bands (C–OH stretching mode and C–O and C–O–C ring vibrations of carbohydrates)[60,63,67,68] and nucleic acid bands (P=O, C–O–P, P–O–P, and P=O symmetric stretching of PO–2).[60,63,67,69]

Table 2

Characteristic Absorption Bands Observed for the Residue of the Biotic Solution (Simulated Marine Solution with SRB)

absorption bands (cm–1)	assignment	assignment reference
2920	CH2 asymmetric stretch	60–66
2850	CH2 symmetric stretch	60–66
∼1640	C=O of amide I	60, 61, and 64–66
1544	N–H and C–N of amide II	60, 61, and 64–66
1454	C–H bend from CH2	60, 61, and 64–66
∼1400	C–O bends from carboxylate ions	60 and 63–66

Though the intensities of the peaks that are detected on the intact two-step silane-coated sample are higher than those of the silane-coated sample pre-immersed in chloride solution with bacteria, similar peaks are observed for both samples. The broad band with a shoulder that appears in the frequency regime of 1200–900 cm–1 is presumably due to the siloxane band (Si–O–Si) as a similar peak with the shoulder is seen in the FTIR spectra of intact silane at the same frequency region. In addition, the peaks corresponding to the amino groups (asymmetric, symmetric, protonated, and deformation) and C–H stretching groups (asymmetric and symmetric) are also detected. Interestingly, the bands of amides I and II at ∼1640 and ∼1540 cm–1 were not detected in the FTIR spectrum of the silane-coated surface. The presence of amide bands in the FTIR spectrum is a typical indication of the bacterial cell presence in bulk solution (as supported by the FTIR spectrum of the residue), suggesting that only a limited number of bacteria could attach on the silane-coated surface; thus, the developed two-step silane coating containing quaternary ammonium silane (QAS) was effective in mitigation of microbial corrosion of mild steel in the marine solution containing bacteria.

SEM Results

SEM micrographs of uncoated and two-step silane-coated steel pre-immersed for 14 days in biotic solution are depicted in Figure . Dense bacterial colonies, the biofilm, and corrosion products are attached to the uncoated steel surface pre-exposed to the biotic solution for 14 days. However, there were considerably less bacterial adhesion and fewer corrosion products deposited on the B(BQ,npH)5:1-coated surface during 14 days of pre-exposure. The bacterial aggregations are limited to a few patchy deposits. Such cluster formation/aggregation is a natural response of bacteria to avoid exposure to toxic materials.[70] The EDX spectra in Figure c,d of the silane-coated steel shows the presence of Si, Fe, C, O, and S. While Fe and S indicate the FeS formation, Si, C, and O correspond to the silane film on the steel surface. The higher intensities of Si, C, and O peaks suggest the presence of the silane film on the steel surface even after 14 days of exposure to marine solution containing SRB.

Figure 9

(a,b) SEM micrographs of the uncoated and two-step silane-coated mild steel pre-exposed to biotic solution for 14 days. (c,d) EDX spectra of the two-step silane-coated mild steel.

Conclusions

The two-step silane coating of BTSE followed by a silane mixture of bis-amino silane and QAS with a mixing ratio of 5:1 (B(BQ,npH)5:1) was deposited on mild steel to inhibit microbial corrosion in the marine environment inoculated with sulfate-reducing bacteria. EIS results suggested that the two-step silane coating containing QAS can considerably mitigate microbial corrosion of the mild steel in the biotic solution. The results showed that the corrosion resistance of the coated steel was about four times higher than that of uncoated steel, both pre-exposed for 7 days to biotic solution. For a longer exposure for 14 days, the impedance was still almost three times higher than that of uncoated steel. Potentiodynamic polarization measurements showed that the corrosion current density of uncoated steel was more than 12 times higher than that of the two-step coated steel after 14 days of pre-exposure to the bacterial marine solution.

There were a significant number of active cells attached to the uncoated mild steel surface pre-exposed to bacterial marine solution as suggested by the MPN test. However, the number of active cells adhering to mild steel decreased significantly after treatment with silane containing the QAS. In ATR-FTIR analysis, the amide peaks (I and II), which are the fingerprint of bacterial cells, were not detected on the silane-treated mild steel specimens pre-exposed to the biotic medium as compared to the residue collected from the biotic solution. SEM observations support the electrochemical and FTIR measurements where much fewer bacteria were seen to have attached to the coated steel compared to the dense bacterial attachment and corrosion products detected on the uncoated specimens.