**1. Introduction**

Microbiologically influenced corrosion (MIC), which is caused by interactions between various microorganisms, is a long-term concern in many industries, including those involved with underground pipelines, storage vessels, shipping, and marine equipment. Reports indicate that MIC was responsible for at least 20% of all damaging corrosion, with a direct cost of \$30–\$50 billion annually worldwide [1,2]. Among the various types of micro-organisms, SRB are considered the major bacterial group responsible for microbial corrosion under anaerobic conditions [3,4]. Several studies have been conducted to investigate SRB-induced corrosion of iron substrate, and SRB are widely accepted to play a crucial role in the anaerobic MIC of iron, low-alloy steel, and stainless steel [5–17]. Various mechanisms explain the enhanced corrosion caused by SRB. Among these, the primary mechanism sees SRB reduce inorganic sulfate to hydrogen sulfide, resulting in the bulk equation (Fe + H2S → FeS + H2) [18–21]. Moreover, extracellular polymer substances and corrosive metabolites secreted by SRB [22,23], consumption of cathodic hydrogen and iron-derived electron transfers [24], and anodic depolarization resulting from the local acidification at the anode [25], all affect the corrosion behavior of iron.

Because of economic losses and safety hazards, it is important to control microbial corrosion by SRB and aggressive sulfide anions when they contact metal substrates. Several methods such as biocide treatment, cathodic protection, and addition of nitrate (or nitrite) have been developed to minimize the risks resulting from SRB activity [23,26]. Among these, biocide treatment is the most common method of controlling microbial corrosion. Organotins, such as dibutyltin and tributyltin (TBT), have been extensively used as antifouling agents in paints since the early 1970s. Despite their high resistance to microbial corrosion in marine environments, these compounds were banned in numerous parts of the world in the early 1990s due to the severe environmental and human health risks that they pose [27–33]. Therefore, nontoxic or less-toxic alternative biocides must be developed to substitute for TBT.

Quaternary ammonium compounds (QACs) are considered candidates for biocides because of their excellent stability and antimicrobial properties [34–36]. QACs are surfactants with a hydrocarbon water-repellent (hydrophobic) group and a water-attracting group (hydrophilic). The antimicrobial properties of QACs mainly depend on the length of the long-chain alkyl group [37]. QACs have wide applications ranging from clinical to industrial purposes. For example, they are used for the disinfection of surfaces, equipment, and medical devices. Benzyldimethyldodecylammonium chloride (BDMDAC) is a quaternary ammonium compound with a C12-alkyl chain. The negative charge of a cell membrane easily attracts the positive charge of the ammonium group in BDMDAC. The hydrophobic C12-alkyl chain inserts into the membrane, causing disruption of bacterial cells [38]. Thus, BDMDAC is regarded as highly bactericidal. Comparing the median lethal dose (LD50) values of BDMDAC with the TBT obtained from their respective material safety datasheets (Alfa Aesar, Ward Hill, MA, USA, 2015), the LD50 value for BDMDAC is two to three times higher than that of TBT (LD50 of BDMDAC: 400 mg/kg in rats; LD50 of TBT: 132 mg/kg in rats), suggesting BDMDAC is less toxic than TBT.

The objective of this study was to evaluate the antimicrobial properties of BDMDAC in anaerobic conditions by examining the effect of BDMDAC as a biocide on *D. desulfuricans*. The influence of BDMDAC and *D. desulfuricans* on the corrosion behavior of 304 stainless steel (304SS) was studied using polarization curves.

#### **2. Materials and Methods**

#### *2.1. Sample Preparation*

The 304SS coupons (2 × 1 × 0.1 cm3) with a testing area of 2 cm<sup>2</sup> were prepared for the subsequent corrosion studies and biofilm observation. All the coupons were mechanically ground using emery papers of 200–1200 grit, rinsed with deionized water, and then washed with alcohol in an ultrasonic bath.

#### *2.2. Bacteria and Culture Medium*

*Desulfovibrio desulfuricans* subsp. *desulfuricans*, the SRB used in this study, was obtained from the Bioresource Collection and Research Center, Hsinchu, Taiwan. The medium used for *D. desulfuricans*, Postgate's medium (DSMZ, *Desulfovibrio* medium, Medium 63), was prepared as follows [39–41]: A 980 mL solution A (0.5 g of K2HPO4, 1 g of NH4Cl, 2 g of MgSO4·7H2O, 1 g of Na2SO4, 0.1 g of CaCl2·2H2O, 1 g of yeas<sup>t</sup> extract, and 2 g of sodium lactate) was boiled, and the dissolved oxygen in the solution was removed using a mechanical deaerator with nitrogen. A 10 mL solution B (0.1 g of ascorbic acid and 0.1 g of sodium thioglycolate) and a 10 mL solution C (0.5 g of FeSO4·7H2O) were added to solution A. The medium was adjusted to pH 7.8 with NaOH and autoclaved at 120 ◦C for 15 min. *D. desulfuricans* was inoculated in the medium and cultured at 37 ◦C.

#### *2.3. Bactericidal Assay of BDMDAC*

Optical density at 600 nm (OD600) was measured using UV/VIS spectrophotometer (Optizen Pop, Mecasys, Daejeon, Korea). The *D. desulfuricans* culture was left overnight and diluted to an OD600

of 0.1 with a fresh medium. Various concentrations of BDMDAC were added to the culture and the absorbance at 600 nm was recorded as bacterial growth curves.

#### *2.4. Electrochemical Measurements*

Potentiodynamic polarization curves were deduced using a Gamry Reference 600 potentiostat (Warminster, PA, USA) to evaluate corrosion performance [5,6,8,42–44]. A standard three-electrode system comprising a graphite counter electrode and saturated calomel electrode (SCE) as a reference electrode was used in all electrochemical tests. Three polarization curve measurements were performed under different working solutions as follows: (1) 304SS coupons in a culture medium without *D. desulfuricans* inoculation and BDMDAC (labeled as a blank solution); (2) 304SS coupons in a *D. desulfuricans*-inoculated medium without BDMDAC (labeled as an SRB solution); and (3) 304SS coupons in a *D. desulfuricans*-inoculated medium with BDMDAC (labeled as a BDMDAC solution). Potentiodynamic polarization curve measurements were obtained by sweeping the potential from −0.5 to 1.5 V versus open circuit potential at a scan rate of 1 mV/s [42]. Corrosion potential (*Ecorr*) and current density (*Icorr*) were determined through Tafel extrapolation. The test area on the 304SS coupons for all electrochemical tests was 2 cm2.

#### *2.5. Surface Characterization*

Biofilm formation and corrosion products formed on the 304SS coupons after different immersion times in various working solutions were analyzed through scanning electron microscopy (SEM) (JSM-6510, JEOL, Tokyo, Japan) and energy-dispersive X-ray spectrum (EDS) (Inca x-act, Oxford Analytical Instruments, Abington, UK). After the immersion tests, the 304SS coupons were extracted, rinsed with distilled water, and then fixed with 2.5 wt % glutaraldehyde for 15 min, followed by dehydration in a graded series of ethanol solutions (30%, 50%, 70%, 90%, and 100% for 15 min each) and air drying [7,45–47]. The dried coupons were coated with plate platinum on the surface and then studied using SEM. EDS was performed to analyze the chemical compositions of the biofilms and corrosion products. The surface morphology of 304SS coupons after polarization curve measurements was examined under an optical microscope (OM) (SG-3006HM, SAGE Vision, New Taipei City, Taiwan).
