# Comparison of 304 SS, 2205 SS, and 410 SS Corrosion by Sulfate-ReducingDesulfovibrio ferrophilus

Authors: Junlei Wang; Hongfang Liu; Pruch Kijkla; Sith Kumseranee; Suchada Punpruk; Magdy El-Said Mohamed; Mazen A. Saleh; Tingyue Gu
Journal: Journal of Chemistry
 (2021)

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## Abstract

Three types of stainless steel (304 SS, 410 SS, and 2205 SS) were evaluated for their corrosion behaviors in microbiologically influenced corrosion (MIC) byDesulfovibrio ferrophilus strain IS5, a relatively new and very corrosive sulfate-reducing bacteria (SRB) strain. The incubation lasted for 7 days in enriched artificial seawater at 28°C and the results showed that 410 SS had a rather large weight loss (6.2 mg/cm2) and a maximum pit depth (118 µm), but 2205 SS and 304 SS did not suffer from significant weight loss or pitting. Electrochemical tests indicated that 2205 SS was slightly more resistant to SRB MIC than 304 SS, while 410 SS was far less resistant.


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## Body

## 1. Introduction

Stainless steel is widely used in various industries. The 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 sulfate-reducing 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 [11–17].Among them, SRB are the main culprits in MIC because sulfate is widely distributed in soil, seawater, and oil and gas systems [12, 18–20]. They 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 byD. 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.


## 2. Materials and Methods

### 2.1. Specimen Preparation

The elemental compositions of the 3 stainless steel types are listed in Table1. 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]. The cleaned coupons were weighed using an analytical balance with a readability of 0.1 mg. The 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.Table 1
Elemental compositions (wt. %) of 3 stainless steel types (Fe balance).
SSCPSSiMnCrNiMoNCuCoAlSn3040.0630.0310.0020.3121.42218.3308.1180.2970.0450.3164100.1290.020.0010.290.4312.240.420.120.0270.050.120.020.00122050.0290.0290.0010.3871.72122.4914.7213.2210.1740.237


### 2.2. Culture Medium Preparation

D. ferrophilus strain IS5 was purchased from a culture collection (DSMZ, Braunschweig, Germany). It was incubated at 28°C in enriched artificial seawater (EASW) for 7 days. The chemical composition of EASW was as follows (in g/L): NaCl 23.476, Na2SO4 3.917, NaHCO3 0.192, KCl 0.664, KBr 0.096, SrCl2·6H2O 0.04, H3BO3 0.026, trisodium citrate 0.5, CaCl2·2H2O 1.469, MgCl2·6H2O 10.61, yeast extract 1, sodium lactate 3.5, NH4Cl 0.1, CaSO4·2H2O 0.1, Fe (NH4)2 (SO4)2·6H2O 1.38, and K2HPO4 0.05. Its initial pH was adjusted to 7.0–7.2 using a 1 M (w/w) NaOH solution before sterilization. The culture medium and tools were sterilized in an autoclave (Model MLS-3751L, Panasonic, Osaka, Japan) for 20 min at 121°C. Then, the EASW was sparged with 99.99% (v/v) N2 for 1 h to remove O2 [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.All anaerobic manipulations were conducted in an anaerobic chamber (Model HE-493, Vacuum Atmosphere Company, Hawthorne, CA, USA) with N2 atmosphere [27]. All chemicals in this work were purchased from either Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).


### 2.3. 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. Then, 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. Then, 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. This method is far more accurate than MPN (most probably number) method to enumerate planktonic and sessile cell counts if individual cells can be seen.


### 2.4. Electrochemical Measurements

A three-electrode electrochemical glass cell setup (Figure1) 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°cm2 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
Three-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. The center glass tube was for the reference electrode.
(a)(b)


## 2.1. Specimen Preparation

The elemental compositions of the 3 stainless steel types are listed in Table1. 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]. The cleaned coupons were weighed using an analytical balance with a readability of 0.1 mg. The 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.Table 1
Elemental compositions (wt. %) of 3 stainless steel types (Fe balance).
SSCPSSiMnCrNiMoNCuCoAlSn3040.0630.0310.0020.3121.42218.3308.1180.2970.0450.3164100.1290.020.0010.290.4312.240.420.120.0270.050.120.020.00122050.0290.0290.0010.3871.72122.4914.7213.2210.1740.237


## 2.2. Culture Medium Preparation

D. ferrophilus strain IS5 was purchased from a culture collection (DSMZ, Braunschweig, Germany). It was incubated at 28°C in enriched artificial seawater (EASW) for 7 days. The chemical composition of EASW was as follows (in g/L): NaCl 23.476, Na2SO4 3.917, NaHCO3 0.192, KCl 0.664, KBr 0.096, SrCl2·6H2O 0.04, H3BO3 0.026, trisodium citrate 0.5, CaCl2·2H2O 1.469, MgCl2·6H2O 10.61, yeast extract 1, sodium lactate 3.5, NH4Cl 0.1, CaSO4·2H2O 0.1, Fe (NH4)2 (SO4)2·6H2O 1.38, and K2HPO4 0.05. Its initial pH was adjusted to 7.0–7.2 using a 1 M (w/w) NaOH solution before sterilization. The culture medium and tools were sterilized in an autoclave (Model MLS-3751L, Panasonic, Osaka, Japan) for 20 min at 121°C. Then, the EASW was sparged with 99.99% (v/v) N2 for 1 h to remove O2 [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.All anaerobic manipulations were conducted in an anaerobic chamber (Model HE-493, Vacuum Atmosphere Company, Hawthorne, CA, USA) with N2 atmosphere [27]. All chemicals in this work were purchased from either Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).


## 2.3. 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. Then, 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. Then, 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. This method is far more accurate than MPN (most probably number) method to enumerate planktonic and sessile cell counts if individual cells can be seen.


## 2.4. Electrochemical Measurements

A three-electrode electrochemical glass cell setup (Figure1) 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°cm2 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
Three-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. The center glass tube was for the reference electrode.
(a)(b)


## 3. Results and Discussion

### 3.1. Cell Counts and Weight Loss

Figure1(a) shows an electrochemical glass cell right after inoculation (day 0). The 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.Table2 shows the planktonic and sessile cells after the 7-day incubation at 28°C. Obviously, 410 SS vial had the largest numbers of both planktonic and sessile cells, but much smaller than one log. The largest sessile cell count in the 410 SS vial was a factor for why 410 SS had the largest corrosion rate (6.2 mg/cm2) 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.Table 2
SRB cell counts after 7-day incubation.
MetalPlanktonic (×106 cells/ml)Sessile (×107 cells/cm2)304 SS1.5 ± 0.11.7 ± 0.2410 SS2.5 ± 0.46.3 ± 0.32205 SS1.8 ± 0.23.5 ± 0.3Table 3
Weight loss after 7-day incubation (standard deviations from replicate coupons in three SRB bottles).
MetalWeight loss (mg/cm2)304 SSUndetectable410 SS6.2 ± 0.22205 SSUndetectable


### 3.2. pH Values

The pH values before and after the 7-day incubation are listed in Table4. 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 CO2 which can cause some acidity. The escape of H2S 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. The 410 SS vials had much more weight loss, contributing to pH increase. Thus, in MIC studies, a shift in pH sometimes can be a predictor of weight loss change:(1)2CH3CHOHCOO−+2H2O⟶2CH3COO−+2CO2+8H++8e−(2)4Fe⟶4Fe2++8e−(3)SO42−+9H++8e−⟶HS−+4H2OTable 4
Broth pH before and after incubation in 125 mL vial.
MetalDay 0Day 7304 SS7.016.75410 SS7.017.132205 SS7.016.79Table2 shows the highest planktonic and sessile cell counts occurred for the vial with 410 SS among the 3 cases. More H2S was likely produced due to better growth.Some researchers blame H2S as behind SRB MIC of Fe(0) based on the following reaction [33]:(4)Fe+H2S⟶FeS+H2.However, H2S corrosion of carbon steel requires a significantly acidic pH as demonstrated in abiotic H2S corrosion research [34]. A telltale sign for H2S 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]. Thus, the corrosion process did not generate hydrogen gas. This effectively ruled out H2S 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. Thus, this type of MIC is known as EET-MIC [21, 24, 32].


### 3.3. IFM

After removing corrosion products and biofilms upon ending the 7-day incubation, the surface morphologies of the stainless steel were examined with results shown in Figure2. In Figure 2, 304 SS and 2205 SS show a smooth surface with polishing lines. However, 410 SS shows serious localized corrosion in Figure 2(b). 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 well-defined 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/cm3. This large pit was easily visible even to the naked eye. Although in the literature a weight loss as high as 5.7 mg/cm2, 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 O2 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 cm2) coupon surface areas. Some replicate 410 SS coupons did not have obvious pits. In this work, they were excluded.Figure 2
Surface images of 3 stainless steel types after 7-day incubation: (a) 304 SS, (b) 410 SS, and (c) 2205 SS. Note: red bar indicates IFM scan path in Figure3(b).Figure 3
Surface profiles of three kinds of stainless steel after 7-day incubation: (a) 304 SS, (b) 410 SS, and (c) 2205 SS.
(a)(b)(c)


### 3.4. EIS

EIS measurements were performed daily. Nyquist plots of the 3 stainless steel types during the 7 days of incubation are shown in Figure4. The 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. This 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 (Rct) 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 Rf + Rct) from day 1 to day 7, indicating that 304 SS was more stable in the SRB environment than 410 SS (Figure 4(c)). The 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. Thus, 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. The 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 4
Nyquist plots of three stainless steel types during 7-day incubation. (a) 0 day. (b) 304 SS. (c) 410 SS. (d) 2205 SS.
(a)(b)(c)(d)Table 5
Fitted electrochemical parameters from EIS data.
DayRs (Ω cm2)Qf (Ω−1 sn cm−2)n1Rf (Ω cm2)Qdl (Ω−1 sn cm−2)n2Rct (Ω cm2)W1–R (Ω cm2)W1–T (s)W1–P304 SS09.21.87 × 10−50.771.60 × 1041.04 × 10−50.761.34 × 106110.82.78 × 10−40.955.468.08 × 10−40.844.63 × 104312.17.80 × 10−40.924.172.56 × 10−30.913.93 × 104510.89.49 × 10−40.944.132.37 × 10−30.904.31 × 104710.79.15 × 10−40.923.632.33 × 10−30.914.80 × 104410 SS08.31.83 × 10−60.98946.22 × 10−60.791.88 × 10618.52.48 × 10−30.89491.10 × 10−40.872.82 × 10437.97.99 × 10−30.861.72×1024.98 × 10−30.972.10 × 1039.66 × 10316.70.6157.98.45 × 10−30.8552.84.86 × 10−20.931.23 × 1022.35 × 10424.80.8277.81.15 × 10−20.8070.71.52 × 10−20.822.52 × 1023.99 × 10338.50.732205 SS07.77.45 × 10−60.844.18×1021.23 × 10−50.771.86 × 10618.32.26 × 10−30.881.49×1021.79 × 10−120.741.70 × 10438.16.84 × 10−30.9464.83.98 × 10−90.757.70 × 10357.96.72 × 10−30.9541.51.01 × 10−80.711.57 × 10477.96.76 × 10−30.9456.01.09 × 10−80.702.24 × 104Figure 5
Equivalent circuit model used for fitting EIS data: (a) all conditions, except for (b) 410 SS on days 3–7.
(a)(b)


### 3.5. OCP

Figure6 shows the OCP tendency with time. OCP of the 3 stainless steel types fluctuated in the first 2 days and then kept relatively stable. The 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. This is consistent with the weight loss data in Table 3.Figure 6
OCP versus time during 7-day incubation.


### 3.6. Linear Polarization Resistance

Figure7 shows the Rp tendency with time. Rp is inversely proportional to corrosion rate. On day 0, 410 SS had the highest Rp before biofilm establishment. After 1 day of incubation, Figure 7 indicates that the 410 SS Rp 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 7
Rp versus time during 7-day incubation.


### 3.7. Potentiodynamic Polarization

Figure8 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 (icorr). The fitted Tafel parameters are listed in Table 6. It shows that the icorr of 410 SS is 21.3 μA/cm2, much larger than the icorr values of 304 SS and 2205 SS. This directly supports the weight loss data in Table 4.Figure 8
Potentiodynamic polarization curves of 3 stainless steel types after 7-day incubation.Table 6
Tafel parameters fitted from potentiodynamic polarization curves after 7 days.
Metalβa (mV/dec)βc (mV/dec)Ecorr (mV) versus SCEicorr (μA/cm2)304 SS70 ± 4−275 ± 9−427 ± 20.495 ± 0.003410 SS257 ± 12−428 ± 11−466 ± 421.3 ± 0.12205 SS105 ± 6−253 ± 5−416 ± 31.38 ± 0.02


## 3.1. Cell Counts and Weight Loss

Figure1(a) shows an electrochemical glass cell right after inoculation (day 0). The 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.Table2 shows the planktonic and sessile cells after the 7-day incubation at 28°C. Obviously, 410 SS vial had the largest numbers of both planktonic and sessile cells, but much smaller than one log. The largest sessile cell count in the 410 SS vial was a factor for why 410 SS had the largest corrosion rate (6.2 mg/cm2) 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.Table 2
SRB cell counts after 7-day incubation.
MetalPlanktonic (×106 cells/ml)Sessile (×107 cells/cm2)304 SS1.5 ± 0.11.7 ± 0.2410 SS2.5 ± 0.46.3 ± 0.32205 SS1.8 ± 0.23.5 ± 0.3Table 3
Weight loss after 7-day incubation (standard deviations from replicate coupons in three SRB bottles).
MetalWeight loss (mg/cm2)304 SSUndetectable410 SS6.2 ± 0.22205 SSUndetectable


## 3.2. pH Values

The pH values before and after the 7-day incubation are listed in Table4. 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 CO2 which can cause some acidity. The escape of H2S 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. The 410 SS vials had much more weight loss, contributing to pH increase. Thus, in MIC studies, a shift in pH sometimes can be a predictor of weight loss change:(1)2CH3CHOHCOO−+2H2O⟶2CH3COO−+2CO2+8H++8e−(2)4Fe⟶4Fe2++8e−(3)SO42−+9H++8e−⟶HS−+4H2OTable 4
Broth pH before and after incubation in 125 mL vial.
MetalDay 0Day 7304 SS7.016.75410 SS7.017.132205 SS7.016.79Table2 shows the highest planktonic and sessile cell counts occurred for the vial with 410 SS among the 3 cases. More H2S was likely produced due to better growth.Some researchers blame H2S as behind SRB MIC of Fe(0) based on the following reaction [33]:(4)Fe+H2S⟶FeS+H2.However, H2S corrosion of carbon steel requires a significantly acidic pH as demonstrated in abiotic H2S corrosion research [34]. A telltale sign for H2S 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]. Thus, the corrosion process did not generate hydrogen gas. This effectively ruled out H2S 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. Thus, this type of MIC is known as EET-MIC [21, 24, 32].


## 3.3. IFM

After removing corrosion products and biofilms upon ending the 7-day incubation, the surface morphologies of the stainless steel were examined with results shown in Figure2. In Figure 2, 304 SS and 2205 SS show a smooth surface with polishing lines. However, 410 SS shows serious localized corrosion in Figure 2(b). 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 well-defined 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/cm3. This large pit was easily visible even to the naked eye. Although in the literature a weight loss as high as 5.7 mg/cm2, 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 O2 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 cm2) coupon surface areas. Some replicate 410 SS coupons did not have obvious pits. In this work, they were excluded.Figure 2
Surface images of 3 stainless steel types after 7-day incubation: (a) 304 SS, (b) 410 SS, and (c) 2205 SS. Note: red bar indicates IFM scan path in Figure3(b).Figure 3
Surface profiles of three kinds of stainless steel after 7-day incubation: (a) 304 SS, (b) 410 SS, and (c) 2205 SS.
(a)(b)(c)


## 3.4. EIS

EIS measurements were performed daily. Nyquist plots of the 3 stainless steel types during the 7 days of incubation are shown in Figure4. The 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. This 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 (Rct) 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 Rf + Rct) from day 1 to day 7, indicating that 304 SS was more stable in the SRB environment than 410 SS (Figure 4(c)). The 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. Thus, 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. The 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 4
Nyquist plots of three stainless steel types during 7-day incubation. (a) 0 day. (b) 304 SS. (c) 410 SS. (d) 2205 SS.
(a)(b)(c)(d)Table 5
Fitted electrochemical parameters from EIS data.
DayRs (Ω cm2)Qf (Ω−1 sn cm−2)n1Rf (Ω cm2)Qdl (Ω−1 sn cm−2)n2Rct (Ω cm2)W1–R (Ω cm2)W1–T (s)W1–P304 SS09.21.87 × 10−50.771.60 × 1041.04 × 10−50.761.34 × 106110.82.78 × 10−40.955.468.08 × 10−40.844.63 × 104312.17.80 × 10−40.924.172.56 × 10−30.913.93 × 104510.89.49 × 10−40.944.132.37 × 10−30.904.31 × 104710.79.15 × 10−40.923.632.33 × 10−30.914.80 × 104410 SS08.31.83 × 10−60.98946.22 × 10−60.791.88 × 10618.52.48 × 10−30.89491.10 × 10−40.872.82 × 10437.97.99 × 10−30.861.72×1024.98 × 10−30.972.10 × 1039.66 × 10316.70.6157.98.45 × 10−30.8552.84.86 × 10−20.931.23 × 1022.35 × 10424.80.8277.81.15 × 10−20.8070.71.52 × 10−20.822.52 × 1023.99 × 10338.50.732205 SS07.77.45 × 10−60.844.18×1021.23 × 10−50.771.86 × 10618.32.26 × 10−30.881.49×1021.79 × 10−120.741.70 × 10438.16.84 × 10−30.9464.83.98 × 10−90.757.70 × 10357.96.72 × 10−30.9541.51.01 × 10−80.711.57 × 10477.96.76 × 10−30.9456.01.09 × 10−80.702.24 × 104Figure 5
Equivalent circuit model used for fitting EIS data: (a) all conditions, except for (b) 410 SS on days 3–7.
(a)(b)


## 3.5. OCP

Figure6 shows the OCP tendency with time. OCP of the 3 stainless steel types fluctuated in the first 2 days and then kept relatively stable. The 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. This is consistent with the weight loss data in Table 3.Figure 6
OCP versus time during 7-day incubation.


## 3.6. Linear Polarization Resistance

Figure7 shows the Rp tendency with time. Rp is inversely proportional to corrosion rate. On day 0, 410 SS had the highest Rp before biofilm establishment. After 1 day of incubation, Figure 7 indicates that the 410 SS Rp 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 7
Rp versus time during 7-day incubation.


## 3.7. Potentiodynamic Polarization

Figure8 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 (icorr). The fitted Tafel parameters are listed in Table 6. It shows that the icorr of 410 SS is 21.3 μA/cm2, much larger than the icorr values of 304 SS and 2205 SS. This directly supports the weight loss data in Table 4.Figure 8
Potentiodynamic polarization curves of 3 stainless steel types after 7-day incubation.Table 6
Tafel parameters fitted from potentiodynamic polarization curves after 7 days.
Metalβa (mV/dec)βc (mV/dec)Ecorr (mV) versus SCEicorr (μA/cm2)304 SS70 ± 4−275 ± 9−427 ± 20.495 ± 0.003410 SS257 ± 12−428 ± 11−466 ± 421.3 ± 0.12205 SS105 ± 6−253 ± 5−416 ± 31.38 ± 0.02


## 4. Conclusion

In this work, three kinds of stainless steel (304 SS, 410 SS, and 2205 SS) were inoculated withD. ferrophilus to evaluate their corrosion behaviors. After 7 days, 304 SS and 2205 SS yielded undetectable weight loss, while 410 SS had a rather large weight loss of 6.2 mg/cm2. The higher sessile cell count for 410 SS contributed to its large weight loss, but the metallurgical properties likely played a much bigger role. 410 SS contains much less Cr and Ni than 304 SS and 2205 SS. Moreover, there was severe pitting on 410 SS (maximum pit depth 118 µm), while well-defined pits were not found on 304 SS and 2205 SS. Electrochemical tests indicated that, at the beginning of the 7-day incubation, 410 SS showed a higher corrosion resistance than 304 SS and 2205 SS, suggesting that 410 SS had a better anticorrosion property before the SRB biofilm was fully established. As time went on, 410 SS showed a lower corrosion resistance than 304 SS and 2205 SS. Corrosion current density (icorr) data at the end of the 7-day incubation indicated that the icorr of 410 SS was 21.3 μA/cm2, 43 times that for 304 SS (0.495 μA/cm2) and 15 times that for 2205 SS (1.38 μA/cm2). This directly supports the weight loss data and pitting data.



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*Source: 3268404-2021-06-17.xml*