Electrochemical Assessment of Mitigation of Desulfovibrio ferrophilus IS5 Corrosion against N80 Carbon Steel and 26Cr3Mo Steel Using a Green Biocide Enhanced by a Nature-Mimicking Biofilm-Dispersing Peptide

Abstract

MIC (microbiologically influenced corrosion) is problematic in many industries, especially in the oil and gas industry. In this work, N80 carbon steel for pipelines was tested with 26Cr3Mo chromium pipeline steel for comparison in SRB (sulfate-reducing bacterium) MIC mitigation using a THPS (tetrakis hydroxymethyl phosphonium sulfate)-based commercial biocide (Biotreat 5475 with 75–80% THPS by mass). Peptide A, a nature-mimicking synthetic cyclic peptide (cys-ser-val-pro-tyr-asp-tyr-asn-trp-tyr-ser-asn-trp-cys) with biofilm dispersal ability was used as a biocide enhancer. Metal coupons covered with 3-d old Desulfovibrio ferrophilus IS5 biofilms were immersed in different biocide solutions. After 1-h treatment, 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM (360 ppb) Peptide A, and 400 ppm Biotreat 5475 achieved 0.5-log, 1.7-log and 1.9-log reductions in sessile cell count on N80, and 0.7-log, 1.7-log, and 1.8-log on 26Cr3Mo, respectively. The addition of 200 nM Peptide A cut the THPS biocide dosage by nearly half. Biocide injection tests in electrochemical glass cells after 1 h exhibited 15%, 70%, and 72% corrosion inhibition efficiency (based on corrosion current density) on N80, and 27%, 79%, 75% on 26Cr3Mo, respectively. Linear polarization resistance and electrochemical impedance spectrometry results also indicated antimicrobial efficacies.

1. Introduction

N80 carbon steel is a common pipeline steel in oilfield applications. Like other carbon steels, N80 is susceptible to different types of corrosion such as CO₂ (carbon dioxide) corrosion and microbiologically influenced corrosion (MIC) [1,2,3,4]. Low chromium steels have much higher corrosion resistance to CO₂ and they are more economical than stainless steels [5,6,7]. The corrosion product films on chromium steels serve as a passive layer to hinder the outward diffusion of Fe²⁺, and the enrichment of Cr prevents corrosive anions from attacking the steel surface [8,9,10]. The addition of Cr in steels also improves MIC through the passive film a certain degree of antimicrobial effect of Cr ions [11]. 26Cr3Mo is a new grade chromium steel that belongs to 3Cr-L80 series pipeline steels [12].

MIC was first identified a century ago [13]. It is a major issue in the oil and gas industry due to its contribution to equipment failures including pipelines, and pressure vessels [14,15,16]. MIC is reported to be responsible for more than 20% of the total corrosion costs [17,18]. Sulfate-reducing bacteria (SRB) are involved in the majority of severe MIC cases. SRBs are ubiquitous in oilfield operations, because sulfate is widely present in many oilfield operations owing to the use of seawater injection in enhanced oil recovery [19,20,21,22]. To mitigate SRB MIC, biocide treatment is a commonly used technique in oilfields [23,24] together with pigging.

SRB MIC of Fe⁰ (elemental iron) is known to belong to extracellular electron transfer-MIC (EET-MIC) in which sessile cells utilize electrons from extracellular Fe⁰ oxidation for intracellular sulfate reduction in energy production [25]. The following half-reactions can be used to analyze the bioenergetics of the SRB corrosion process with sulfate serving as the terminal electron acceptor [25].

4Fe → 4Fe²⁺ + 8e⁻ (E° = −447 mV_SHE)

(1)

SO₄²⁻ + 9H⁺ + 8e⁻ → HS⁻ + 4H₂O (E°’ = −217 mV_SHE)

(2)

The combined reaction has a positive cell potential of +230 mV (at 1 M solutes except H⁺, and pH 7 as indicated by the apostrophe in E). This means this thermodynamically favored corrosion process generates energy that can benefit SRB metabolism. More sessile cells harvest more electrons from Fe⁰. Numerous studies demonstrated that in SRB MIC of Fe⁰, there was a direct correlation showing more sessile cells corresponding to higher weight loss [1]. Thus, in EET-MIC, treating biofilms to reduce the sessile cell counts is the key to MIC mitigation.

When metabolite-MIC (M-MIC) is involved, a biofilm such as an acid-producing bacteria (APB) biofilm can produce local high acidity because volumetric sessile cell density is much higher than planktonic cell density. Thus, mitigating M-MIC also requires biofilm treatment.

Biofilms provide embedded sessile cells with protection from antimicrobial agents [26,27,28]. A much higher dosage of biocide is needed to treat sessile cells compared with planktonic cells [29,30,31]. As a result, traditional biocide dosing suffers from high operational costs and potential environmental issues [32,33]. A mixed-culture field biofilm can become more resistant to a certain biocide after using it for a period of time, because the killing of vulnerable microbes leaves a nutritional niche for more resistant microbes to move in from the surrounding environment [34,35,36]. This leads to a decrease of biocide efficacy and a demand for higher dosages to mitigate the biofilms [37,38].

Environmental-friendly biocides with minimal or no toxicity are preferred in mitigating MIC. Tetrakis hydroxymethyl phosphonium sulfate (THPS) is a popular green biocide in oilfield operations [39,40]. THPS degrades to trihydroxymethyl phosphine [41], which can effectively reduce disulfide bonds in disulfide amino acids in microbial cell walls, leading to cleavage of the bonds and destruction of the cell walls [42,43]. The selective action of THPS can also affect the hNRB (heterotrophic nitrate-reducing bacteria) and so-NRB (sulfide-oxidizing-NRB) activities, resulting in SRB growth inhibition and preventing sulfide formation [44]. Other than killing or inhibiting SRB, THPS can scavenge H₂S (hydrogen sulfide) in a chemical reaction to mitigate souring [45].

In field operations, green biocide enhancers can be added to biocides to improve biocide efficacy as well as to minimize environmental impact. A small amount of biocide enhancer can significantly improve biocide efficacy or lower the biocide dosage [37]. It was reported that 50 ppm D-amino acid mixtures enhanced 15 ppm THPS to achieve a similar efficacy as 30 ppm THPS against an oilfield biofilm consortium in a lab test [46]. Peptide A is a chemically synthesized 14-mer circular peptide (cys-ser-val-pro-tyr-asp-tyr-asn-trp-tyr-ser-asn-trp-cys). Its core 12-mer sequence was inspired by a biofilm-dispersing protein secreted by a sea anemone to maintain its biofilm-free exterior [47]. Peptide A was reported to be an effective non-biocidal green biocide enhancer against an oilfield biofilm consortium [48]. It was found that in MIC of the oilfield biofilm consortium on C1018 carbon steel, the combination of 100 ppm DBNPA (2,2-dibromo-3-nitrilopropionamide) + 100 nM Peptide A achieved 0.9-log, 0.8-log and 0.6-log further reductions in sessile cell count for SRB, APB (acid-producing bacteria) and GHB (general heterotrophic bacteria), respectively, compared to treatment with 100 ppm DBNPA alone [47]. It was reported that Peptide A alone was not biocidal at or below 100 nM dosage, but when it is used together with a biocide, it exhibited a biofilm dispersal effect which enhanced the biocide efficacy [48].

Chromium steels provide good passivation against CO₂ corrosion. However, their passive films are (semi-)conductive, allowing an SRB biofilm to harvest electrons across the passive film. Once there is a defective or damaged passive film spot, the spot will be preferentially used for Fe²⁺ outward diffusion in a large cathode-small anode scenario, leading to classical pitting or pit amplification [49]. It was found that after a 7-d incubation with Desulfovibrio ferrophilus, 13%Cr had a weight loss of 4.4 mg/cm² (0.30 mm/a uniform corrosion rate) while its maximum pit depth reached 288 μm (15 mm/a pitting rate), compared to a much larger weight loss of 15.2 mg/cm² (1.0 mm/a uniform corrosion rate) accompanied by a much lower maximum pit depth of 7.3 μm (0.38 mm/a pitting rate) for N80 which had much inferior FeS passivation to the passivation provided by Cr oxides/hydroxides on 13%Cr [49]. This means that to deploy a Cr steel for CO₂ corrosion resistance applications, an MIC assessment and mitigation plan should be in place because there is potential for severe MIC attack.

Before a biocide is field tested, lab testing is desired to probe dosage and efficacy. There are two kinds of biocide tests. One is the biofilm prevention test, in which treatment chemicals are added to the culture medium upon inoculation [48]. The other is the biofilm kill (or biofilm eradication) test that uses biocides to treat pre-established biofilms [46,50,51]. In pipelines, the kill test is relevant, in which a biocide liquid “plug” moves downstream between two pigs driven by pressure. The residence time is rather short. In lab tests, such a short-term (e.g., 0.5 h or 1 h) will not generate a measurable weight loss or pit depth difference, unlike in biofilm prevention tests that last for days. Thus, real-time or near-real-time corrosion rate measurements are needed to calculate corrosion rate changes for the calculation of biocide corrosion inhibition efficiency that can be used to support biofilm sessile cell count reduction. Only electrochemical tests can provide near-real-time transient corrosion rate measurements. OCP (open circuit potential), LPR (linear polarization resistance), EIS (electrochemical impedance spectroscopy), and PDP (potentiodynamic polarization) scans are commonly used electrochemical techniques to assess corrosion [52,53,54].

This work investigated the mitigation efficacy of a THPS-based biocide, namely Biotreat 5475, enhanced by Peptide A against SRB MIC on N80 and 26Cr3Mo. The sessile cell reductions after a 1-h biocide treatment, and electrochemical responses to biocide injection were examined. Because sessile cells in a mature biofilm are more difficult to treat than preventing a biofilm from establishing on a metal surface, relatively high dosages (200 ppm and 400 ppm) of Biotreat 5475 were employed.

2. Results and Discussion

2.1. Enumeration of Sessile Cells on Coupons Soaked in Petri Dishes with and without Biocide

The anaerobic vials containing N80 and 26Cr3Mo coupons before and after the 3-d incubation are shown in Figure 1. All the vials turned black due to FeS precipitation, indicating healthy SRB growth. Figure 2 demonstrates sessile cell count results on N80 and 26Cr3Mo after the 1-h biofilm kill test (soaking test). The initial sessile cell count on N80 after the 3-d incubation was (5.5 ± 0.3) × 10⁷ cells/cm². After the 1-h treatment with 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM Peptide A, and 400 ppm Biotreat 5475, the sessile cell counts were reduced to (1.8 ± 0.4) × 10⁷ cells/cm², (1.2 ± 0.4) × 10⁶ cells/cm², and (7.3 ± 1.3) × 10⁵ cells/cm², respectively. Thus, 200 ppm Biotreat 5475 only achieved a 0.5-log reduction in sessile cells, while 200 ppm Biotreat 5475 + 200 nM Peptide A, and 400 ppm Biotreat 5475 achieved 1.7-log and 1.9-log sessile reduction. The difference between 200 ppm Biotreat 5475 + 200 nM Peptide A and 400 ppm Biotreat 5475 was not statistically significant (p value = 0.25 >> 0.05). Thus, 200 nM Peptide A achieved an enhancement effect of extra 1.2-log sessile cell reduction. It effectively lowered the THPS dosage from 400 ppm to 200 ppm.

For 26Cr3Mo, the initial sessile cell count found after the 3-d incubation was (4.4 ± 0.5) × 10⁷ cells/cm². After the 1-h treatment with 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM Peptide A, and 400 ppm Biotreat 5475, sessile cell counts were reduced to (9.8 ± 2.2) × 10⁶ cells/cm², (8.8 ± 1.8) × 10⁵ cells/cm², and (7.5 ± 2.0) × 10⁵ cells/cm², respectively. These reductions in sessile cells were equivalent to 0.7-log, 1.7-log, and 1.8-log, respectively. For 200 ppm Biotreat 5475 + 200 nM Peptide A and 400 ppm Biotreat 5475, there was no significant statistical difference (p value = 0.57 >> 0.05). Thus, 200 nM Peptide A achieved an enhancement effect of extra 1.0-log sessile cell reduction, and it cut THPS dosage by nearly half. Sessile cells on 26Cr3Mo were slightly fewer than those on N80 under the same conditions, which can be explained by the inhibition of microbial growth due to some alloying metal elements such as Cr, Mo in 26Cr3Mo [55].

2.2. Electrochemical Test Results

OCP responses of N80 and 26Cr3Mo during the 1 h after the biocide injection treatment are shown in Figure 3 and Figure 4. Without biocide injection, there were very few variations in OCP for N80 and 26Cr3Mo (controls). After biocide injection, OCP values of both N80 and 26Cr3Mo exhibited a decreasing trend over the 1-h period. This is obviously misleading. Theoretically, a lower OCP value indicates a greater corrosion tendency (working electrode losing electrons). However, in most SRB MIC cases, OCP trends are wrong because OCP only indicates corrosion tendency, not the actual corrosion kinetic process or corrosion outcome [47]. Kinetic electrochemical measurements which have been repeatedly proven reliable should be depended upon instead [25,48,52].

Figure 5 and Figure 6 show the polarization resistance (R_p) variation during the 1-h biocide treatment. Because the inverse of R_p from LPR can be used to represent corrosion rate, the decrease of 1/R_p can estimate corrosion inhibition efficacy for a biocide treatment,

$${\mathsf{\eta }}_{\mathrm{i},\mathrm{LPR}}=\left(1-\frac{{\mathrm{R}}_{\mathrm{p},\mathrm{o}}}{{\mathrm{R}}_{\mathrm{p}}}\right)\times 100\%$$

(3)

In the no-biocide injection control glass cell, there were only small variations in R_p. After biocide injection, R_p values of N80 and 26Cr3Mo gradually increased, suggesting corrosion rate reduction due to biocide treatment. After the 1-h treatment, 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM Peptide A, and 400 ppm Biotreat 5475 achieved 24%, 31%, and 43% 1/R_p reductions, respectively for N80. For 26Cr3Mo, the reductions were 34%, 41%, and 40%, respectively. The cocktail of 200 ppm Biotreat 5475 + 200 nM Peptide A led to an extra 1/R_p reduction for both N80 and 26Cr3Mo compared to 200 ppm Biotreat 5475 alone.

The equivalent electrical circuit model shown in Figure 7 was used for EIS spectra modeling. R_s and R_f are solution resistance and resistance of the film consisting of the biofilm and the corrosion products, respectively. R_ct denotes charge transfer resistance. Q_f and Q_dl stand for the capacitance of the biofilm/corrosion product film and the double-layer capacitance, respectively. Nyquist and Bode plots of N80 and 26Cr3Mo are displayed in Figure 8 and Figure 9. The Nyquist plots in Figure 8A–D and Figure 9A–D indicate that biocide treatment increased the semi-circle diameters, indicating increased corrosion resistance. The EIS fitting results are listed in

Table 1. Fitted electrochemical parameters of N80 from EIS data in Figure 8.

	t (h)	Rs (Ω cm2)	Qf (Ω−1 sn cm−2)		n1	Rf (Ω cm2)	Qdl (Ω−1 sn cm−2)	n2	Rct (Ω cm2)	Rf + Rct (Ω cm2)
No treatment	0	11.5	4.1 × 10−2	0.41		0.28	8.6 × 10−2	0.79	82.0	82.0
	1	10.5	4.9 × 10−2	0.44		0.25	9.3 × 10−2	0.80	86.0	86.0
200 ppm Biotreat 5475	0	5.52	7.2 × 10−2	0.98		1.69	0.12	0.55	54.1	55.8
	1	5.16	0.22	0.79		15.2	0.18	0.64	80.0	95.2
200 ppm Biotreat 5475 + 200 nM Peptide A	0	6.32	0.10	0.55		0.17	6.0 × 10−2	0.98	63.7	63.9
	1	5.98	0.14	0.80		42.3	0.43	0.83	109	151
400 ppm Biotreat 5475	0	6.24	9.6 × 10−2	0.79		44.9	0.87	0.98	18.9	63.8
	1	6.01	0.16	0.81		31.4	0.27	0.70	134	165

and

Table 2. Fitted electrochemical parameters of 26Cr3Mo from EIS data in Figure 9.

	t (h)	Rs (Ω cm2)	Qf (Ω−1 sn cm−2)	n1	Rf (Ω cm2)	Qdl (Ω−1 sn cm−2)	n2	Rct (Ω cm2)	Rf + Rct (Ω cm2)
No treatment	0	5.38	9.9 × 10−3	0.49	1.66	0.27	0.87	128	130
	1	5.59	6.5 × 10−3	0.53	1.93	0.20	0.85	140	142
200 ppm Biotreat 5475	0	10.2	8.0 × 10−2	0.89	36.9	0.13	0.61	84.9	122
	1	8.46	7.1 × 10−2	0.87	44.1	0.12	0.65	119	163
200 ppm Biotreat 5475 + 200 nM Peptide A	0	15.3	1.3 × 10−6	0.39	12.1	0.32	0.72	133	145
	1	15.8	0.25	0.69	0.01	8.2 × 10−13	0.79	277	277
400 ppm Biotreat 5475	0	9.73	0.35	0.77	88.6	3.9 × 10−3	0.61	1.37	90
	1	9.87	0.26	0.70	222	0.16	0.76	0.01	222

.

(R_ct + R_f) is often used to estimate corrosion resistance that is comparable to R_p [56,57]. Because its inverse can represent corrosion rate, 1/(R_ct + R_f) decrease is used to estimate corrosion inhibition efficacy for a biocide treatment,

$${\eta }_{\mathrm{i},\mathrm{EIS}}=\left(1-\frac{R_{\mathrm{ct},0}+R_{\mathrm{f},0}}{R_{\mathrm{ct}}+{\mathrm{R}}_{\mathrm{f}}}\right)\times 100\%$$

(4)

In Figure 10 and Figure 11, (R_ct + R_f) was stable for N80 and 26Cr3Mo during the 1-h period without biocide injection. One h after the biocide injection, (R_ct + R_f) of both N80 and 26Cr3Mo increased significantly, suggesting decreased corrosion rates due to biocide injection. Based on fitted EIS parameters in Table 1 and Table 2, for N80, the 1-h biocide treatment with 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM Peptide A, and 400 ppm Biotreat 5475 reduced 1/(R_ct + R_f) by 41%, 58%, and 61%, respectively. For 26Cr3Mo, the reductions were 25%, 28%, and 60%, respectively. The EIS results indicated that the efficiency sequence was 200 ppm Biotreat 5475 < 200 ppm Biotreat 5475 + 200 nM Peptide A < 400 ppm Biotreat 5475, which is consistent with LPR results.

Figure 12 and Figure 13 present Tafel curves of N80 and 26Cr3Mo after biocide treatment with Tafel scans starting 1.5 h after the biocide injection. The fitted Tafel parameters are summarized in

Table 3. Tafel parameters of N80 fitted from potentiodynamic polarization curves in Figure 12.

	βa (V/dec)	βc (V/dec)	Ecorr (V) vs. SCE	icorr (mA/cm2)
No treatment	0.103	−0.440	−0.611	0.54
200 ppm Biotreat 5475	0.205	−0.287	−0.645	0.46
200 ppm Biotreat 5475 + 200 nM Peptide A	0.161	−0.148	−0.649	0.16
400 ppm Biotreat 5475	0.200	−0.192	−0.656	0.15

and

Table 4. Tafel parameters of 26Cr3Mo fitted from potentiodynamic polarization curves in Figure 13.

	βa (V/dec)	βc (V/dec)	Ecorr (V) vs. SCE	icorr (mA/cm2)
No treatment	0.191	−0.271	−0.647	0.26
200 ppm Biotreat 5475	0.208	−0.301	−0.675	0.19
200 ppm Biotreat 5475 + 200 nM Peptide A	0.286	−0.242	−0.677	0.055
400 ppm Biotreat 5475	0.287	−0.245	−0.659	0.066

. A higher corrosion current density (i_corr) correlates to a higher uniform corrosion rate. Without biocide injection, the i_corr values were 0.54 mA/cm² and 0.26 mA/cm² for N80 and 26Cr3Mo. A lower i_corr value of 26Cr3Mo also suggests that 26Cr3Mo had a greater (uniform) corrosion resistance than N80.

Corrosion inhibition efficiency (η_i) calculated from i_corr is shown in

Table 5. Biocide efficacy data from sessile cell reduction, and estimated MIC inhibition efficiencies from various electrochemical tests.

			Corrosion Inhibition Efficiency
	Sessile Cell Count Reduction		LPR 1/Rp Reduction (ηi,LPR)		EIS 1/(Rct + Rf) Reduction (ηi,EIS)		PDP icorr Reduction (ηi)
	N80	26Cr3Mo	N80	26Cr3Mo	N80	26Cr3Mo	N80	26Cr3Mo
200 ppm Biotreat 5475	68.2%	78.0%	24%	34%	41%	25%	15%	27%
200 ppm Biotreat 5475 + 200 nM Peptide A	97.8%	98.0%	31%	41%	58%	48%	70%	79%
400 ppm Biotreat 5475	98.7%	98.3%	43%	40%	61%	60%	72%	75%

. After the 1-h biocide treatment, η_i values for N80 were 15%, 70%, and 72% for 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM Peptide A, and 400 ppm Biotreat 5475, respectively. For 26Cr3Mo, the values were 27%, 79%, and 75%, respectively. The 200 ppm Biotreat 5475 didn’t achieve a high corrosion inhibition, but 200 nM peptide turned out to enhance the biocide efficacy considerably, reaching a similar corrosion inhibition outcome as 400 ppm Biotreat 5475 did, which means cutting THPS dosage by half, consistent with the trend in sessile cell counts.

Table 5 summarizes the biocide efficacy data from sessile cell reduction, LPR 1/R_p reduction, EIS 1/(R_ct + R_f) reduction, and PDP i_corr reduction. The data in this work indicated that all three electrochemical methods correctly supported sessile cell reduction outcomes. Table 5 data indicate that biocide treatment efficacies were similar for the two metals. Although different electrochemical methods provided different corrosion inhibition efficacy values, their trends were consistent. Thus, LPR, EIS, and PDP were all able to support the sessile cell count trend.

The findings in this work were comparable to other Peptide A studies in that Peptide A turned out to be an effective biocide enhancer that was able to cut the biocide dosage by nearly half [47,48,52]. Peptide A was found attractive as it functioned at a very low concentration (sub-ppm level). The biofilm dispersal effect appeared crucial in preventing biofilm formation and MIC mitigation. The mechanism of the biofilm dispersal effect of Peptide A was speculated in a previous study. It will be worthwhile to investigate it in depth in the future.

3. Materials and Methods

3.1. Bacterium, Metal, and Chemicals

Table 6. Elemental compositions (wt. %) of N80 [59] and 26Cr3Mo (Fe balance).

Metal	C	Mn	Cr	Ni	Cu	P	S	Si	Mo	V
N80	0.34–0.38	1.45–1.70	0.15			0.020	0.015	0.20–0.35
26Cr3Mo	0.25	0.49	2.99	0.02	0.02	0.008	0.001	0.26	0.12	0.015

shows the elemental compositions of 26Cr3Mo and N80 steels. Biotreat 5475 was an industrial biocide from Clariant (Muttenz, Switzerland) that contained 75–80% of THPS by mass. Peptide A with 97.2% purity (based on peak area analysis in reverse-phase high-performance liquid chromatography) according to the supplier (Bachem Holding AG, Bubendorf, Switzerland) was used in this work.

Table 7. Test matrix for 1-h SRB biofilm kill test in Petri dishes after 3-d pre-growth.

Parameter	Condition
Microbe	D. ferrophilus IS5
Cultural medium	EASW
Coupon material	N80 and 26Cr3Mo
Liquid volume	50 mL in 125 mL anaerobic vials
Inoculum size	0.5 mL in anaerobic vials
Treatment method	No treatment control 200 ppm Biotreat 5475 200 ppm Biotreat 5475 + 200 nM Peptide A 400 ppm Biotreat 5475 1 cm2 coupon surface/25 mL liquid
Temperature	28 °C
Initial pH	7.0 ± 0.2
Incubation time before biocide soaking	3 d
Biocide treatment time	1 h in Petri dish in an anaerobic chamber
Assay	Sessile cell counts on coupons

and

Table 8. Test matrix for electrochemical responses to biocide injection into 3-d SRB broth.

Parameter	Condition
Microbe	D. ferrophilus IS5
Cultural medium	EASW
Coupon material	N80 and 26Cr3Mo
Liquid volume	250 mL in 450 mL glass cells
Inoculum size	2.5 mL in glass cells
Treatment method	No treatment control 200 ppm Biotreat 5475 200 ppm Biotreat 5475 + 200 nM Peptide A 400 ppm Biotreat 5475 4 glass cells used. Injection was followed by 3 min gentle shaking to dispere biocide.
Temperature	28 °C
Initial pH	7.0 ± 0.2
Incubation time before biocide injection	3 d
Biocide treatment time	1 h
Assay	OCP, LPR, EIS, and Tafel scans using glass cells

display the test matrices for this work. D. ferrophilus (strain IS5), a highly-corrosive pure strain SRB [49,58], was immersed in enriched artificial seawater (EASW) culture medium inoculated with D. ferrophilus at 28 °C for 3 days to yield mature biofilms on the metal coupon surfaces. The composition of EASW is listed in

Table 9. Composition of EASW culture medium [52].

Chemical	Amount
NaCl	23.476 g
Na2SO4	3.917 g
NaHCO3	0.192 g
KCl	0.664 g
KBr	0.096 g
H3BO3	0.026 g
MgCl2	4.965 g
SrCl2·6H2O	0.04 g
CaCl2·2H2O	1.469 g
Sodium lactate	3.5 g
Yeast extract	1.0 g
Sodium citrate	0.5 g
MgSO4·7H2O	0.71 g
CaSO4·2H2O	0.1 g
NH4Cl	0.1 g
K2HPO4	0.05 g
Fe(NH4)2(SO4)2·6H2O	1.37 g
DI water	1 L

. The pre-cut 26Cr3Mo and N80 coupons were coated with an inert liquid Epoxy coating (3M Product 323) except for a 10 mm × 10 mm exposed top work surface. The coupons were left at room temperature for drying overnight. The top surface was polished to 600 grit and the coupon was sterilized with anhydrous isopropanol before testing. The initial pH of EASW was adjusted to 7.0 using 5% (w/w) NaOH. The medium was sterilized in an autoclave at 121 °C for 20 min. Then, it was sparged with filter-sterilized N₂ for 1 h to get rid of dissolved oxygen. Finally, an oxygen scavenger L-cysteine was added into the medium to reach a final concentration of 100 ppm. The biocide stock solution was filter-sterilized using a 0.22 μm Stericup single-use sterile filter (Millipore, Bedford, MA, USA). The inoculation (with a 1:100 volume ratio for inoculum vs. culture medium) was carried out in an anaerobic chamber filled with N₂.

3.2. Sessile Cell Counts

For counting sessile cells, coupons of each metal type were put into separate 125 mL anaerobic vials with 50 mL EASW. After the 3-d pre-growth of D. ferrophilus, all coupons were taken out in the anaerobic chamber and rinsed with pH 7.4 phosphate-buffered saline (PBS) to remove planktonic cells. The coupons were then transferred into Petri dishes containing the pH 7.4 PBS solutions with and without biocide chemicals (1 cm² coupon surface per 25 mL PBS) in the anaerobic chamber. After the 1-h biocide soaking treatment, sessile cells, which were motile, were counted using a hemocytometer under a 400× microscope [25].

3.3. Electrochemical Measurements

Electrochemical tests were performed in 450 mL glass cells, each filled with 250 mL EASW. For each metal type (N80 or 26Cr3Mo), 4 glass cells were used (1 for no-biocide control, 3 for 3 different biocide treatments). A three-electrode setup was adopted for measurements (Figure 14A). The working electrode (WE) Epoxy cake contained two replicate coupons as replicates (Figure 14B). The counter electrode (CE) was a thin platinum sheet, and a saturated calomel electrode (SCE) was used as the reference electrode (RE). Electrochemical measurements were carried out using a PCI4/750 potentiostat (Gamry Instruments, Inc., Warminster, PA, USA). OCP, LPR, EIS and PDP were measured in this work. LPR was measured at a scanning rate of 0.167 mV/s from −10 mV to 10 mV (vs. OCP) after OCP became stable. EIS was scanned from 10⁵ Hz to 0.01 Hz with a 10 mV amplitude sinusoidal signal. PDP was measured at the end of incubation. Tafel curves were obtained from two half-scans on the same working electrode starting from 0 mV to −200 mV (vs. OCP) and 0 mV to +200 mV (vs. OCP) with a scanning rate of 0.167 mV/s.

In the electrochemical tests, D. ferrophilus biofilms were pre-grown on the working electrode surfaces to reach maturity, which took 3 days of incubation. After that, a concentrated biocide stock solution was injected into the anaerobic glass cell. The glass cell was then gently shaken for 3 min to disperse the biocide. OCP and LPR were measured every 20 min during the 1 h following the biocide injection. EIS scan was conducted just before and after the 1-h biocide treatment to evaluate the biofilm’s response to the biocide treatment. Tafel scans were performed after other electrochemical measurements were performed. To evaluate corrosion inhibition efficiency (η_i), reduction in i_corr was calculated using the following equation [52]:

$${\eta }_{\mathrm{i}}=\left(1-\frac{i_{\mathrm{corr}}}{i_{\mathrm{corr},0}}\right)\times 100\%$$

(5)

where i_corr and i_corr,0 represent corrosion current densities with and without biocide treatment, respectively. i_corr,0 was obtained in the biotic control glass cell.

The biofilm kill test by soaking coupons in a biocide solution simulates a concentrated biocide plug between two pigs moving down a pipeline. Please note that corrosion weight loss change and corrosion pit depth change in this kind of 1-h (to simulate 1 h contact time or residence time) biofilm kill test are not measurable. Electrochemical tests are the only methods that can provide near-real time corrosion rate changes for biocide efficacy assessment to support sessile cell count reduction data.

4. Conclusions

26Cr3Mo showed a higher uniform corrosion resistance compared to N80 under the same conditions based on R_p and i_corr values before biocide treatment. The SRB biofilms on both metals responded similarly to Biotreat 5475 treatment. The various electrochemical corrosion measurements indicated that 200 ppm Biotreat 5475 was able to mitigate MIC by D. ferrophilus, but it was not very effective. The addition of 200 nM Peptide A considerably enhanced the efficacy of 200 ppm Biotreat 5475, which achieved similar efficacy as 400 ppm Biotreat 5475. This indicates that the addition of 200 nM Peptide cut the THPS dosage by nearly half. This work demonstrated that LPR, EIS, and PDP are reliable in assessing antimicrobial efficacy. They provided corrosion rate information that supported sessile cell reductions after biocide treatment in the 1-h biofilm kill test in which weight loss and pit depth changes were not measurable.