Sulfate-Reducing Desulfovibrio ferrophilus

Department of Chemical and Biomolecular Engineering, Institute for Corrosion and Multiphase Technology, Ohio University, Athens, OH 45701, USA School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China PTT Exploration and Production, Bangkok 10900, 0ailand Research and Development Center, Saudi Arabian Oil Company, Dhahran 31311, Saudi Arabia


Introduction
Stainless steel is widely used in various industries. e typical austenitic stainless steel such as 304 SS is widely used in pipelines and power plants because of its good ductility, wear resistance, and corrosion resistance [1,2]. Classic martensitic stainless 410 series have been widely used in petroleum production and machine parts, exhibiting high strength and hardness but being less resistant to corrosion compared to other types of stainless steel [3]. For instance, 410 SS is the most popular martensitic grade SS. 2205 SS is duplex stainless steel with a mixture of austenitic and delta-ferritic phases [4]. It is widely used in heat exchanger pipes, pressure vessels, and marine engineering [5,6]. Duplex stainless steel has demonstrated that its corrosion resistance in these media is similar to that of traditional austenitic types or even better [7].
Microbiologically influenced corrosion (MIC) is one of the main causes of metal corrosion [8,9]. Twenty percent of corrosion costs is attributed to MIC, leading to a great economic loss every year based on reports [10]. MIC is widespread in the oil and gas industry, marine systems, and water cooling systems, involving different kinds of bacteria. Various microorganisms such as bacteria including sulfatereducing bacteria (SRB) and acid-producing bacteria (APB), iron-oxidizing bacteria (IOB), nitrate-reducing bacteria (NRB), fungi, and methanogens (archaea) have been reported to lead to serious corrosion by their metabolic activities in different environments [11][12][13][14][15][16][17].
Among them, SRB are the main culprits in MIC because sulfate is widely distributed in soil, seawater, and oil and gas systems [12,[18][19][20]. ey can utilize sulfate as the terminal electron acceptor and organic carbon as electron donor [21,22]. Dinh et al. isolated a specialized and novel SRB strain with a much higher corrosion ability known as Desulfovibrio ferrophilus strain IS5. It can utilize elemental iron as the sole and direct electron donor for its metabolism [23]. D. ferrophilus reduces sulfate using Fe as an electron donor much faster than traditional hydrogen consumption SRB species [23]. It is interesting to investigate its corrosion against various stainless steel.
In this work, three types of stainless steel (304 SS, 2205 SS, and 410 SS) were tested for their resistance against MIC by D. ferrophilus incubated in enriched artificial seawater. Planktonic and sessile cell counts were used to describe microbial growth. Weight loss and pit depth were used to quantify corrosion severity. Electrochemical measurements were used to corroborate weight loss and pitting data trends and to provide transient corrosion behavior.

Specimen Preparation.
e elemental compositions of the 3 stainless steel types are listed in Table 1. Stainless steel coupons with a 1 cm × 1 cm unpainted top area were used for corrosion testing. Before testing, coupons were abraded by 180-, 400-, and 600-grit emery papers sequentially, then cleaned with 100% isopropanol, and sterilized under an UV lamp for 30 min [24]. After 7 days of incubation, coupons were taken out. Corrosion products and the biofilm on each coupon surface were removed using Clarke's solution [25]. e cleaned coupons were weighed using an analytical balance with a readability of 0.1 mg. e coupon surfaces were scanned using an infinite focus microscope (IFM) (Model ALC13, Alicona Imaging GmbH, Graz, Austria) to analyze pitting corrosion. An optical microscope at 400x was used to visualize pits on coupons. 0.05. Its initial pH was adjusted to 7.0-7.2 using a 1 M (w/w) NaOH solution before sterilization. e culture medium and tools were sterilized in an autoclave (Model MLS-3751L, Panasonic, Osaka, Japan) for 20 min at 121°C. en, the EASW was sparged with 99.99% (v/v) N 2 for 1 h to remove O 2 [26]. A L-cysteine stock solution was sterilized using a 0.22 μm sterile filter (Stericup, Millipore, Bedford, MA, USA). It was added to the culture medium to reach 100 ppm (w/w) as an oxygen scavenger.

Culture Medium
All anaerobic manipulations were conducted in an anaerobic chamber (Model HE-493, Vacuum Atmosphere Company, Hawthorne, CA, USA) with N 2 atmosphere [27]. All chemicals in this work were purchased from either Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).

Planktonic and Sessile Cell Counts.
Each 125 mL vial contained 3 coupons in 100 mL EASW plus 1 mL inoculum with roughly 23 mL headspace. After the 7-day incubation at 28°C, coupons were taken out and rinsed in a pH 7.4 phosphate-buffered solution (PBS) 3 times to remove loosely attached planktonic cells and culture medium. Sessile cells on a coupon were removed using a small disposable brush into a 10 mL PBS solution in a disposable plastic weighing dish. en, the brush, the coupon, and 10 mL PBS solution were vortexed together in a 50 mL conical tube for 30 s. For planktonic cell counting, 1 mL broth from a 125 mL anaerobic vial was first diluted with 9 mL PBS buffer in a 50 mL conical tube. en, this tube was vortexed for 30 s before the diluted cell suspension was counted using a hemocytometer. In this work, a hemocytometer was used to measure planktonic and sessile cell count liquid suspensions under an optical microscope at 400x magnification. is method is far more accurate than MPN (most probably number) method to enumerate planktonic and sessile cell counts if individual cells can be seen.

Electrochemical Measurements.
A three-electrode electrochemical glass cell setup ( Figure 1) was used for electrochemical measurements. Each glass cell was filled with 100 mL EASW with a headspace of 25 mL. A stainless steel coupon sealed in epoxy with an exposed area of 1°cm 2 served as the working electrode. Platinum sheet and saturated calomel electrode (SCE) served as counter and reference electrode, respectively [28]. During the 7-day D. ferrophilus incubation, open circuit potential (OCP), linear polarization resistance (LPR), and electrochemical impedance spectroscopy (EIS) were measured daily. Potentiodynamic polarization (PDP) was performed at the end of 7th day. All electrochemical tests were conducted using a VersaSTAT 3 potentiostat (Princeton Applied Research, Oak Ridge, TN, USA). When OCP became stable, LPR were measured from −10 mV to 10 mV (vs. OCP) with a scanning rate of 0.167 mV/s [29]. EIS were conducted from 105 Hz to 0.01 Hz with a 10 mV AC amplitude [12,30,31]. PDP was measured at the end of incubation. To scan from OCP to 200 mV one working electrode was used, and from OCP to −200 mV, another working electrode was used in a different glass cell [11]. EIS was analyzed using Zview software (version 2). LPR and PDP were analyzed using Cview software (version 2). Figure 1(a) shows an electrochemical glass cell right after inoculation (day 0). e broth darkened after the 7-day incubation (Figure 1(b)) due to FeS precipitation, which proves that D. ferrophilus grew well and produced a lot of FeS. Table 2 shows the planktonic and sessile cells after the 7day incubation at 28°C. Obviously, 410 SS vial had the largest numbers of both planktonic and sessile cells, but much smaller than one log. e largest sessile cell count in the 410 SS vial was a factor for why 410 SS had the largest corrosion rate (6.2 mg/cm 2 ) in Table 3. SRB MIC of SS belongs to EET-MIC (extracellular electron transfer MIC) [21,32]. More sessile cells on the metal surface could take more electrons from the metal, which led to more serious 410 SS corrosion. More importantly, 410 SS has much less Cr and Ni than 304 SS and 2205 SS (Table 1). It is known to be less resistant to abiotic corrosion compared to the other two types of stainless steel [3]. In comparison, 304 SS and 2205 SS had no detectable weight loss.

pH Values.
e pH values before and after the 7-day incubation are listed in Table 4. pH was affected by SRB metabolism and corrosion. In the typical SRB metabolism, lactate oxidation in Reaction (1) coupled with sulfate reduction in Reaction (3) does not produce acidity [22]. On the other hand, lactate oxidation produces CO 2 which can cause some acidity. e escape of H 2 S to the headspace actually takes away some protons from the culture medium, leading to a slight increase in broth pH [22]. More important, was the fact that iron oxidation in Reaction (2) coupled with sulfate reduction in Reaction (3) consumes protons. e 410 SS vials had much more weight loss, contributing to pH increase. us, in MIC studies, a shift in pH sometimes can be a predictor of weight loss change: Figure 1: ree-electrode electrochemical glass cells at day 0 (a) and at the end of 7-day incubation (b). Note: the left wire was for the counterelectrode and the right wire for the working electrode. e center glass tube was for the reference electrode.    Table 2 shows the highest planktonic and sessile cell counts occurred for the vial with 410 SS among the 3 cases. More H 2 S was likely produced due to better growth.
Some researchers blame H 2 S as behind SRB MIC of Fe(0) based on the following reaction [33]: However, H 2 S corrosion of carbon steel requires a significantly acidic pH as demonstrated in abiotic H 2 S corrosion research [34]. A telltale sign for H 2 S corrosion is hydrogen evolution, which is a very sensitive indicator in a sealed vial. It was found that, for an SRB culture, the hydrogen gas concentration profiles in the headspaces of sealed anaerobic vials were the same with and without carbon steel coupons [24]. us, the corrosion process did not generate hydrogen gas. is effectively ruled out H 2 S corrosion as a contributor to SRB MIC of carbon steel near neutral pH. It was sessile cells' harvest of energy from elemental iron via extracellular electron transfer (EET) that is behind the MIC.
us, this type of MIC is known as EET-MIC [21,24,32].  Figure 3 shows coupon surface IFM profiles support this observation, showing a large maximum pit depth of 118 µm for 410 SS, while it was hard to identify welldefined pits in the IFM profiles for 304 SS and 2205 SS surfaces. It was also hard to notice any corrosion pits under a light microscope at 400× in Figure 2. Figure 2 shows that 118 µm pit on 410 SS has a surface diameter of 1 mm. Assuming that the pit had a cylinder shape, the weight loss for the cylinder alone would be 0.72 mg based on 410 SS density of 7.74 g/cm 3 . is large pit was easily visible even to the naked eye. Although in the literature a weight loss as high as 5.7 mg/cm 2 , which is close to that for 410 SS in this work, has been reported for carbon steel MIC by a pure-strain SRB in 7 days [35], the 118 µm pit depth obtained in this work for 410 SS is easily a record in pit depth for any pure-strain SRB MIC of stainless steel with strict O 2 control, suggesting a superior ability of D. ferrophilus for pitting corrosion. It should be noted that severe 410 SS pitting had a random nature in short-term tests using small (1 cm 2 ) coupon surface areas. Some replicate 410 SS coupons did not have obvious pits. In this work, they were excluded.

EIS.
EIS measurements were performed daily. Nyquist plots of the 3 stainless steel types during the 7 days of incubation are shown in Figure 4. e corresponding fitted parameters are listed in Table 5 for the equivalent circuit models shown in Figure 5. Figure 5(b) is used to fit days 3, 5, and 7 in Figure 4(c). All other EIS curves were fitted using Figure 5(a). On day 0 in Figure 4(a), the diameter of the semicircle of 410 SS is the largest and that of 304 SS is the smallest. is means before biofilms were established on the coupon surfaces, 410 SS and 2205 SS were more corrosion resistant than 304 SS. As SRB started to grow, the diameters decreased sharply for all the 3 stainless steel types. Charge transfer resistance (R ct ) decreased by 102 (Table 5), indicating SRB metabolic activities had a large effect on stainless steel corrosion. Among the 3 stainless steel types, 304 SS (Figure 4(b)) and 2205 SS (Figure 4(d)) had much larger corrosion resistance (in terms of R f + R ct ) from day 1 to day 7, indicating that 304 SS was more stable in the SRB environment than 410 SS (Figure 4(c)). e sessile cell count on 410 SS was only 3.7 times larger than 304 SS. However, it was corroded much more than the sessile cell increase could explain.
us, the alloying components in 410 SS played a big role. Table 1 shows that it has much less Cr and Ni than 304 SS and 2205 SS. e diffusion process after day 3 indicated by 410 SS was attributed to the build-up of corrosion products, which required a Warburg impedance element in Figure 5(b) to deal with the mass transfer resistance. Figure 6 shows the OCP tendency with time. OCP of the 3 stainless steel types fluctuated in the first 2 days and then kept relatively stable. e large fluctuation in the first 2 days could be attributed to the SRB cell attachment. After 3 days of incubation, the 410 SS OCP curve is much lower than those for 304 SS and 2205 SS, indicating much higher thermodynamic tendency for 410 SS corrosion.

OCP.
is is consistent with the weight loss data in Table 3. Figure 7 shows the R p tendency with time. R p is inversely proportional to corrosion rate. On day 0, 410 SS had the highest R p before biofilm establishment. After 1 day of incubation, Figure 7 indicates that the 410 SS R p curve is much higher than those of 304 SS and 2205 SS after the first-day transient period, supporting the weight loss data once again. Figure 8 shows the potentiodynamic polarization curves of the 3 stainless steel types performed after 7 days of incubation. It is obvious to see that 410 SS was corroded much more severely than the other two stainless steel types. Both the cathode and anode curves shift to the right, leading to a much larger corrosion current density (i corr ). e fitted Tafel parameters are listed       in Table 6. It shows that the i corr of 410 SS is 21.3 μA/cm2, much larger than the i corr values of 304 SS and 2205 SS. is directly supports the weight loss data in Table 4.

Conclusion
In

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.