Table 1 shows a test matrix for this investigation. D. ferrophilus (strain IS5) was cultured anaerobically in enriched artificial seawater (EASW) culture medium at 28°C (optimal temperature for growth) (Enning et al., 2012). The composition of EASW is listed in Table 2. The test coupon was 13Cr, and its elemental composition is summarized in Table 3.

The EASW culture medium was adjusted to an initial pH of 7.0 using 5% (w/w) NaOH. Before inoculation, the medium was autoclave-sterilized and deoxygenated by purging filter-sterilized N₂ gas for 1 h. The addition of 100 ppm (w/w) L-cysteine into EASW was conducted in an anaerobic chamber filled with N₂ to further remove dissolved oxygen, following its use in the ATCC 1249 culture medium. THPS (Sigma-Aldrich, St. Louis, MO, USA) was dissolved into a 5,000 ppm (w/w) stock solution for biocide treatment.

The pre-cut 13Cr coupons all had an exposed top surface area of 1 cm² (1 cm × 1 cm). The top test surfaces were polished progressively to a 600 grit final finish. All other coupon surfaces were painted with inert Epoxy coating (3M Product 323). The coupons were sanitized using absolute isopropanol before testing.

Three replicate 13Cr coupons were put into each anaerobic vial for different treatment conditions. The initial D. ferrophilus planktonic cell concentration in each vial upon inoculation was 10⁶ cells/mL. After incubation for 7 days, all coupons were taken out from the anaerobic vials. Deoxygenated phosphate buffered saline (PBS) solution (pH 7.4) was then used to rinse the coupons. Sessile cells on each coupon surface were transferred into a 10 mL (or 1 mL if cell count too low for counting in a 10 mL cell suspension) PBS solution (total) using a sterile brush applicator. The brush, the coupon, and the PBS solution were vortexed for 30 s in a 50 mL centrifuge tube. The evenly distributed cell suspension in the tube was used for cell counting. A hemocytometer was employed in which (viable motile) cells can be counted under an optical microscope at ×400 magnification.

Biofilms on coupon surfaces after 7-day incubation were observed under CLSM (confocal laser scanning microscopy) (Model LSM 510, Carl Zeiss, Jena, Germany). Coupons were first rinsed in pH 7.4 PBS solution to wash away planktonic cells and culture medium. Then the coupon surfaces were stained with Live/Dead^® BacLight™ Bacterial Viability Kit L7012 (Life Technologies, Grand Island, NY, United States) before observation. Under CLSM, live cells were seen as green dots at 488 nm wavelength laser and dead cells as red dots at a wavelength of 559 nm.

Three replicate 13Cr coupons were used for each treatment condition in the weight loss test. After the 7-day incubation in 125 mL anaerobic vials, the biofilm and corrosion products on each coupon were removed with a freshly prepared Clarke’s solution for 30 s before weight measurement following ASTM G1–03. Coupon surfaces were scanned using an infinite focus microscope (IFM) profilometer (Model ALC13, Alicona Imaging GmbH, Graz, Austria) to obtain pit depth and surface profiles.

Electrochemical tests were executed in 10 mL electrochemical glass cells, each filled with 5 mL EASW inoculated with 0.05 mL seed culture. Each glass cell consisted of a 13Cr WE (working electrode) (1 cm × 1 cm) and a graphite rod (0.64 cm diameter and 1 cm height) counter electrode, which also served as reference electrode. Electrochemical scans were performed using a PCI4/750 potentiostat (Gamry Instruments, Inc., Warminster, PA, United States). Linear polarization resistance (LPR) and Tafel were scanned daily during the 7-day incubation period. LPR measurement was performed within the range of −10 to 10 mV vs. OCP (open circuit potential) with a rate of 0.167 mV/s. Tafel curves were scanned at a scan rate of 0.167 mV/s from OCP to −200 mV vs. OCP, and then after a 20 min gap, from OCP to +200 mV vs. OCP on the same working electrode (Wang et al., 2021a; Xu et al., 2023b). It was shown that this dual half-scan scheme with the voltage range did not alter the working electrode in repeated scans on the same working electrode (Wang et al., 2022a).

Figure 1 shows images of 125 mL anaerobic vials before and after the 7-day incubation. D. ferrophilus growth was seen in vials with no treatment, 50 ppm THPS and 50 ppm THPS + 100 nM Peptide A. With 100 ppm THPS, no apparent planktonic growth was observed (not turbid), suggesting strong inhibition effect. Table 4 summarizes sessile cell counts on coupons with different treatment.

Without biocide treatment, sessile cells on 13Cr coupons were found to be (3.9 ± 0.8) × 10⁷ cells/cm². With treatment of 50 ppm THPS and 50 ppm THPS + 100 nM Peptide A, sessile cell counts reduced to (8.0 ± 0.4) × 10⁶ cells/cm² and (2.4 ± 0.4) × 10⁶ cells/cm². A 0.7-log reduction was seen in sessile cells with 50 ppm THPS alone, and the addition of 100 nM Peptide A achieved an extra 0.5-log reduction, showing its obvious enhancement effect. Sessile cells were not detected (detection threshold of 5 × 10⁴ planktonic cells/mL translating into 5 × 10⁴ sessile cells/cm²) on coupons treated with 100 ppm THPS.

CLSM images of D. ferrophilus biofilms on 13Cr coupon surfaces with different treatments are presented in Figure 2. Healthy biofilms were seen on the coupon surface without treatment (Figure 2A) with densely populated green dots (live cells). With 50 ppm THPS and 50 ppm THPS + 100 nM Peptide A (Figures 2B, C), live cells became fewer and more dead cells (red dots) appeared. It is hard to see any live cells in Figure 2D on the 13Cr coupon surface treated with 100 ppm THPS. Here, these CLSM biofilm observations are consistent with the sessile cell count results.

The weight loss results of 13Cr coupons with different treatment are listed in Table 5. For the abiotic control, no weight loss was detected. The weight losses for coupons with no treatment, 50 ppm THPS, 50 ppm THPS + 100 nM Peptide A and 100 ppm THPS were 3.8 ± 0.5 mg/cm² (equivalent to uniform corrosion rate 0.26 ± 0.03 mm/a), 2.2 ± 0.2 mg/cm² (0.15 ± 0.01 mm/a), 1.5 ± 0.5 mg/cm² (0.10 ± 0.03 mm/a) and 0.3 ± 0.2 mg/cm² (0.02 ± 0.01 mm/a), respectively. THPS at 50 ppm reduced weight loss by 42%, while 50 ppm THPS + 100 nM Peptide A achieved an extra 18% weight loss reduction, suggesting considerable enhancement effect. With 100 ppm THPS, the uniform corrosion inhibition efficiency reached 92% which was very effective. In EET-MIC by SRB, sessile cells in biofilms are directly involved in iron oxidation. The weight loss sequence matches the sessile cell count sequence. Thus, the greatly reduced weight loss here can be attributed to the effectiveness of THPS in inhibiting the growth of D. ferrophilus biofilms and the biofilm dispersing effect of Peptide A.

The outstanding performance of THPS in mitigating SRB MIC was because the degradation of THPS forms trihydroxymethyl phosphine which can destroy microbial cell walls by reducing disulfide bonds within cell wall disulfide amino acids (Parker and Kharasch, 1959; Rüegg and Rudinger, 1977). THPS also inhibits SRB growth through its selective action which affects the hNRB (heterotrophic nitrate-reducing bacteria) and so-NRB (sulfide-oxidizing-NRB) activities (Okoro, 2015). Besides, as an H₂S scavenger, THPS can mitigate corrosion caused by souring (Talbot et al., 2000). The biofilm dispersing effect of Peptide A enhanced the THPS treatment effect as some sessile cells were detached without biocide kill. The exact mechanism for Peptide A’s superior biofilm dispersing ability remains unknown, but it likely functions as a signal molecule. Thus, its effective dosage is extremely small, much lower than the 10s of ppm dosage required for a typical corrosion inhibitor that needs to have a monolayer coverage on a metal surface.

Coupons with biofilms and corrosion products removed are shown in Figures 3A–E. Some corrosion pits on the coupons are visible to the naked eyes, especially for coupons without treatment in Figure 3B. Figure 4 presents pits with maximum depth and pit profiles scanned using IFM. No well-defined pits were found on the abiotic coupon surface. The maximum pit depth for coupons with no treatment, 50 ppm THPS, 50 ppm THPS + 100 nM Peptide A and 100 ppm THPS were 375 μm (equivalent to 20 mm/a pitting rate), 232 μm (12 mm/a), 163 μm (8.6 mm/a) and 28 μm (1.5 mm/a), respectively. The single deepest pit on the coupon without treatment alone results in a weigh loss of 1.95 mg, accounting for 42% of the entire coupon weight loss. The addition of 50 ppm THPS, 50 ppm THPS + 100 nM Peptide A and 100 ppm THPS reduced pitting rates by 38%, 57%, and 93%, respectively. Thus, 100 ppm THPS mitigated pitting corrosion greatly.

Based on the uniform corrosion rates from weight loss data and pitting corrosion rates, RPS were calculated and summarized in Table 6. The RPS for 13Cr in all conditions were all considerably high but similar with an average value of 80. In comparison, RPS of C1018 carbon steel MIC by D. ferrophilus in EASW was only 1.36 (Wang et al., 2021a). This phenomenon supported the fact that pitting corrosion is the major issue for low grade stainless steels just like 13Cr. Even with biocide treatment, the RPS values showed no apparent changes, which were still almost two orders of magnitude of that for C1018 carbon steel. With 100 ppm THPS, the uniform corrosion rate declined to a very small number of 0.02 mm/a. However, its pitting rate was still as high as 1.5 mm/a, which is greater than pitting rates of some carbon steels in the same system without any biocide treatment (Wang et al., 2021a; Xu et al., 2023b). Thus, a higher THPS dosage may be needed for better biocide mitigation to prevent pinhole leaks due to SRB MIC pitting.

The high PRS values in Table 6 for 13Cr shows that it is particularly prone to pitting corrosion. 13Cr is a low-grade stainless steel (420 SS) with a PREN (pitting resistance equivalent number) value of 13. It has a passive film that is relatively easy to be damaged in some spots compared to higher grade stainless steels such as 304 SS with a PREN value of 20 (L. Xu et al., 2023b). CrSs have Cr oxide/hydroxide passive films that are (semi-)conductive (Sun et al., 2016). In SRB MIC, the iron sulfide corrosion product film is also (semi-)conductive (Gu et al., 2019). Thus, 13Cr MIC by SRB satisfies the two essential requirements for classical pitting, i.e., a passive film that can be damaged and the passive film is (semi-)conductive (L. Xu et al., 2023b). This leads to a large cathode to small anode area ratio situation, which amplifies the pitting effect as illustrated in Figure 5. In carbon steel MIC by SRB, the corrosion products (iron sulfides) form a conductive film that can reach 10–30 μm in thickness (Jia et al., 2018). In stainless steel MIC by SRB, the iron sulfides film will be on top of the metal’s own passive film (chromium oxides/chromium hydroxide) that is only several nm in thickness (Mohammadi et al., 2011). Sessile cells in the biofilm can harvest electrons across both (semi-)conductive films.

Electrochemical tests can provide extra evidence, especially transient information, to support one-shoot weight loss data. Figure 6 demonstrates R _p (polarization resistance) variations of 13Cr with different treatment from LPR measurements. The abiotic control exhibited a stable curve over the 7-day incubation. Without treatment, R _p declined considerably from 1 day to 3 days and remained stable for the remaining time. This suggests that mature biofilm formed at 3 days and corrosion rate generally increased during the first 3 days and stabilized from 3 days to 7 days. With 50 ppm THPS, the R _p curve was consistently higher than that of no treatment. Compared with 50 ppm THPS alone, R _p values of 50 ppm THPS + 100 nM Peptide A were generally higher, especially from 5 days to 7 days. Thus, the enhancement effect of Peptide A for THPS was clearly manifested. The R _p values of 100 ppm THPS did not drop considerably compared to the abiotic control and remained high during the 7-d incubation period, indicating effective corrosion inhibition through biofilm inhibition. The 1/R _p sequence of different treatments matches the corrosion rate sequence based on weight loss results. Based on the 1/R _p data at 7 days, 100 ppm THPS achieved a corrosion inhibition efficiency of 97%.

Corrosion current density (i _corr) results fitted from 7-day Tafel scans are displayed in Figure 7. Without treatment, i _corr gradually increased and remained higher than the treated conditions. With 100 nM Peptide A, i _corr values of 100 ppm THPS were generally lower than those without over the entire 7-day incubation period. The 100 ppm THPS i _corr kept at a low level which was close to the abiotic control, proving that 100 ppm THPS was effective in biofilm inhibition. The i _corr sequence of different treatments again matches the weight loss sequence. Based on the i _corr data at 7 days, 100 ppm THPS achieved a corrosion inhibition efficiency of 93%. It should be noted that daily Tafel scans of the same WE were permitted using the dual-scan method with cathodic scan and cathodic scan commencing at OCP according to our previous Tafel scan scheme study (Wang et al., 2022a). Both LPR and Tafel findings confirmed the effective mitigation of D. ferrophilus MIC on 13Cr and the obvious enhancement effect of Peptide A for THPS. They provide extra evidence to support weight loss or uniform corrosion results. The overall corrosion rates were substantially reduced with biocide addition which also resulted in much lower pitting rates as reflected by the pitting analysis.

Injection tests were conducted in a 10 mL electrochemical cell at 3 days when mature D. ferrophilus biofilms formed on the 13Cr WE surface. In Figure 8, stable R _p readings were obtained for 40 min before injections. After 20 ppm (final concentration in the broth) MNPs injection, a generally decreasing R _p trend was observed and R _p was reduced by 16% in 40 min after the injection, which means the corrosion rate became higher. This shows that MNPs successfully enhanced MIC of D. ferrophilus against 13Cr. THPS was subsequently injected to reach 100 ppm in broth and led to an increase in R _p. The final R _p value was increased by 37% due to the THPS injection, indicating mitigation effect of THPS.

The 16% R _p decreased after the injection of MNPs was because MIC of 13Cr by D. ferrophilus belongs to EET-MIC where the electron transfer between sessile cell walls and Fe surface is an important rate-limiting process, and the MNPs can serve as electron conduits to accelerate this process (Wang et al., 2022b). Thus, the SRB MIC of 13Cr is proven to be EET-MIC here. The followed THPS injection proved its biocidal effect in treating SRB biofilms and showed that electrochemical injection tests were sensitive enough to provide both accelerated and decelerated MIC information. It should be noted that treating a pre-established biofilm is far more difficult than preventing the biofilm from establishing. Thus, 37% was much smaller than the 93% based on the i _corr data at 7 days in Figure 7.