Mitigation of microbial degradation of X80 carbon steel mechanical properties using a green biocide

doi:10.21203/rs.3.rs-2980413/v1

Most microbiologically influenced corrosion (MIC) studies focus on the threat of pinhole leaks caused by MIC pitting. However, microbes can also lead to structural failures. Tetrakis hydroxymethyl phosphonium sulfate (THPS) biocide mitigated the microbial degradation of mechanical properties of X80 pipeline steel by Desulfovibrio ferrophilus, a very corrosive sulfate reducing bacterium. It was found that 100 ppm (w/w) THPS added to the enriched artificial seawater (EASW) culture medium before incubation resulted in approximately 3-log reduction in sessile cell count after a 7-d incubation at 28^oC, leading to 94% weight loss reduction. The X80 dogbone coupon incubated with 100 ppm THPS for 7 d suffered only 3% loss in ultimate tensile strain and 0% loss in ultimate tensile strength compared with the abiotic control in EASW. In comparison, the no-treatment X80 dogbone suffered losses of 13% in ultimate tensile strain and 6% in ultimate tensile stress, demonstrating very good THPS efficacy.

Microbial degradation of mechanical properties

Microbiologically influenced corrosion

Sulfate-reducing bacteria

green biocide

biofilm

Microbial corrosion widely exists in nature and various industrial environments, such as oil and gas systems, water treatment systems, and cooling water systems in power plants, leading to billions of US dollars in losses per year(Ashassi-Sorkhabi et al., 2012; Batmanghelich et al., 2017; Jin et al., 2022; Liu et al., 2022; Wei et al., 2019; Xu et al., 2023a). Facilities in the ocean are more corroded than those in terrestrial environments, and they are difficult to repair (Li et al., 2021b; Lu et al., 2023a; Lu et al., 2023b; Melchers, 2005; Zen, 2005). In the marine environment, microorganisms can adhere to metal surfaces, causing microbiologically influenced corrosion (MIC) that damages engineering facilities and even seriously threatens operators’ safety (Chen et al., 2022; Li et al., 2021a; Li et al., 2022; Xu et al., 2023a; Xu et al., 2022b) in addition of pinhole leaks (Jacobson, 2007).

Most MIC studies focused on MIC pinhole leaks (Chandra et al., 2019; Dao et al., 2021). More attention should be paid to deterioration of mechanical properties of steels, including reductions of tensile strength and strain (Li et al., 2021a; Li et al., 2022). Such deteriorations can lead to catastrophic structural failures if not mitigated. Among the microbes that can cause serious MIC, SRB (sulfate reducing bacteria) accounted for the largest number of severe corrosion cases (Anandkumar et al., 2016; Lu et al., 2023b; Su et al., 2022). D. ferrophilus (strain IS5) is by far the most corrosive marine SRB. It led to a 1.6 mm/a uniform corrosion rate against C1018 carbon steel and 1.5 cm/year pitting rate against 13%Cr steel (i.e., 420 stainless steel) (Wang et al., 2021a; Xu et al., 2023b).

An effective way to reduce the degradation of mechanical properties caused by MIC is to mitigate the biofilms that are behind the MIC, and to reduce the amounts of destructive metabolites. There are many measures to mitigate MIC, such as physical, chemical, and electrochemical methods. They include the addition of alloying elements to steels, scrubbing, anti-corrosion coatings, cathodic protection, and biocides (Li et al., 2023; Videla, 2002; Wang et al., 2021b; Yin et al., 2018). Among these, biocides with broad-spectrum bactericidal effects are the mainstream treatment in mitigating biofilms and MIC (Kahrilas et al., 2015; Sanchez et al., 2014; Wang et al., 2019).

Therefore, in this study, tetrakis hydroxymethyl phosphonium sulfate (THPS), a popular green biocide commonly used in the oil and gas industry as well as water utilities (Okoro et al., 2016; Silva et al., 2021; Xu et al., 2022a), was used to mitigate the microbial degradation of mechanical properties of X80 pipeline steel caused by D. ferrophilus. Bacterial sessile cell count, corrosion surface morphology, corrosion products, and X80 mechanical properties were analyzed to study the efficacy of THPS.

The test matrix for mitigating D. ferrophilus MIC deterioration of X80 mechanical properties using THPS is listed in Table 1. Each coupon was painted with Epoxy resin leaving 1 cm² of exposed surface on each square coupon and 4 cm² total exposed surface area (4 sides in the middle section) of each 76 mm long standard X80 dogbone coupon (ASTM E8, 2013). After coating, the coupons were cleaned using 75% (v/v) isopropanol and dried under a UV light for 20 min.

Table 1

Test matrix for mitigating *D. ferrophilus* MIC deterioration of mechanical properties of X80 steel using THPS biocide
Parameter	Condition
Bacterium	D. ferrophilus (strain IS5)
Culture medium	EASW (composition in Reference(Wang et al., 2021a))
Coupon	X80 square coupon and X80 dogbone coupon
Temperature	28 ˚C
Incubation time	7 d
Anaerobic bottle volume	450 mL
Culture medium volume	300 mL (plus 3 mL seed culture)
Biocide	THPS
Treatment	No treatment (0 ppm THPS) 100 ppm THPS
Analysis	CLSM biofilm imaging, sessile cell count, planktonic cell count, gas measurements, weight loss, pit depth, surface examination using SEM, XPS, and tensile testing

D. ferrophilus (strain IS5) (DSM 15579) in this work was originally isolated from an enrichment culture in North Sea, Wilhelmshaven, Germany according to its vendor (German Collection of Microorganisms and Cell Cultures) (Dinh et al., 2004). An enriched artificial seawater (EASW) culture medium (Wang et al., 2021a) was deoxygenated using N₂ sparging before it was used to incubate D. ferrophilus at 28^oC. Each anaerobic bottle (450 mL) contained 300 mL of deoxygenated EASW, 3 mL D. ferrophilus seed culture, 3 square coupons, and 1 dogbone coupon (leaning against the wall at a 30° angle with respect to the bottom). One anaerobic bottle received no treatment and another received 100 ppm THPS added to EASW upon inoculation. The anaerobic bottles with no treatment and with 100 ppm THPS are shown in Fig. 1.

After a 7-d incubation, live and dead sessile cells on the X80 square coupons were examined using CLSM (confocal laser scanning microscopy) (Chen et al., 2021). The planktonic and sessile cells were enumerate using a Neubauer hemocytometer under a 400× microscope (Cheng et al., 2021). This method works well for motile cells like D. ferrophilus despite their small size. The concentrations of H₂S and H₂ in the headspace and the total gas pressure in each anaerobic bottle were measured at the end of the 7-d incubation as described elsewhere (Jia et al., 2018; Ning et al., 2015).

A freshly prepared Clarke’s solution was used to remove biofilms and corrosion products before weighing each coupon adhering to ASTM G1-03 (Lu et al., 2023b). Pit depths were scanned using a different CLSM machine (VK-X250K, Keyence, Osaka, Japan).

A scanning electron microscope (SEM) machine (FEI Quanta 250, Hillsboro, OR, USA) was employed to examine biofilm morphology on each coupon (Cui et al., 2020). An IFM (InfiniteFocus microscope) profilometer (ALC13, Alicona Imaging GmbH, Graz, Austria) was used to scan for MIC pits on the coupons and to measure corrosion pit depth. An X-ray photoelectron spectroscope (XPS) machine (AXIS Supra, Shimadzu, Kyoto, Japan) was used to analyze the chemical composition of the corrosion products on each coupon. Before XPS analysis, the top biofilm layer on a coupon was removed using a cotton swab to reveal the bottom corrosion products.

After the 7-d incubation, X80 dogbone coupons were tensile tested on a Model E44.304 electromechanical universal testing machine (MTS system, MN, USA). The applied strain rate was 0.004 s^− 1 following literature (Eleiche et al., 1985).

Biofilm imaging, cell counts, and gas measurements

As shown in Fig. 2, on the X80 square coupons incubated with 100 ppm THPS and 0 ppm THPS, the D. ferrophilus sessile cell counts were 1.5×10⁶ cells cm^− 2 and 9.3×10⁸ cells cm^− 2, respectively. The corresponding values on X80 dogbone surfaces were 1.6×10⁶ cells cm^− 2 and 1.1×10⁹ cells cm^− 2 for 100 ppm THPS and 0 ppm THPS, respectively. Thus, the two sets of sessile cell counts were quite close, suggesting that the square coupons could be used to obtain MIC data to save dogbone coupons. The sessile cell count data showed an approximately 3-log reduction with 100 THPS treatment compared to the no-treatment control. As for the planktonic cells, the cell count without THPS was 6.5×10⁸ cells mL^− 1. In the presence of 100 ppm THPS, the planktonic cell count was 1.1×10⁵ cells mL^− 1, a reduction of approximately 3-log was observed (Fig. 3). Thus, 100 ppm THPS offered excellent efficacy for inhibiting both planktonic cells and sessile cells (Jia et al., 2019).

The CLSM images of the biofilms on the X80 square coupons after the 7-d incubation are displayed in Fig. 4. Without THPS, many live sessile cells (green dots) are seen on the coupon. However, in the presence of 100 ppm THPS, the coupon primarily shows dead sessile cells (red dots). This indicates that 100 ppm THPS was very effective for D. ferrophilus biofilm prevention. The same dosage was found effective against D. vulgaris biofilm on C1018 carbon steel, which guided the dosage selection in this work (Wang et al., 2022). The CLSM images here supported the sessile cell counts in Fig. 3.

Table 2 lists the environmental parameters of the anaerobic bottles with and without THPS treatment. With no treatment and 100 pm THPS treatment, the headspace gas pressures were found to be 1.03 bar and 1.01 bar, respectively after the 7-d incubation. The H₂S concentrations were 450 ppm in the headspace, and 4.3×10^− 5 M in the broth (based on Henry’s law calculation (Jia et al., 2018; Ning et al., 2015)) with no treatment, while the 100 ppm THPS treatment yielded roughly 10X lower values of 40 ppm and 3.7×10^− 6 M, respectively.

Table 2

Headspace gas data and broth pH in anaerobic bottles containing *D. ferrophilus* in EASW with and without 100 ppm THPS after 7-d incubation.
D. ferrophilus broth	Total pressure in headspace (bar)	H₂S in headspace (ppm) (v/v)	[H₂S] in broth (M)	Broth pH
100 ppm THPS No treatment	1.01 1.03	40 450	3.7 × 10⁻⁶ 4.3 × 10⁻⁵	7.0 6.9

In SRB metabolism, lactate is first oxidized to pyruvate. Pyruvate is then oxidized to yield acetate with H₂ production. H₂ is used for sulfate reduction (Burns et al., 2012), but some of it escapes to the headspace. When there is a shortage of lactate, some headspace H₂ is consumed as electron donor for sulfate reduction (Smith et al., 2019; Xu et al., 2023a):

CH₃CHOHCOO^– (Lactate) + 2H₂O → CH₃COO^– (Acetate) + 2.5H₂ + HCO₃^– (1)

SO₄ ^2– (Sulfate) + 5H₂ → H₂S + 4H₂O (2)

Weight loss, pit depth, SEM, and XPS analyses

Figure 5 shows the weight loss of the X80 square coupons after the 7-d incubation. The weight losses of the X80 square coupons with and without the 100 ppm THPS treatment were 1.1 mg cm^− 2 (vs. 0.43 mg cm^− 2 weight loss for abiotic control), and 19.5 mg cm^− 2 (equivalent to 1.3 mm/a uniform corrosion rate), respectively. This reflects a 94% THPS treatment efficiency in terms of uniform corrosion reduction.

The pits on the X80 square coupons after the removal of the corrosion products and biofilms are shown in Fig. 6. No obvious pits are observed when 100 ppm THPS was in EASW. In contrast, the coupon with no treatment had a maximum pit depth of 13.5 µm (0.70 mm/a pitting rate) and a maximum pit surface diameter of 36.7 µm. The weight loss and pit depth data with and without 100 ppm THPS in EASW indicate that MIC on the X80 square coupons was greatly reduced by the biocide treatment.

The rather severe MIC uniform corrosion (1.3 mm/a) on the coupon with no THPS treatment occurred because of a robust D. ferrophilus biofilm (9.3×10⁸ cells/cm²) formation on the X80 square coupon. D. ferrophilus sessile cells in the biofilm harvested electrons for respiration in the SRB cytoplasm via extracellular electron transfer (EET) because the electron donor Fe(0) is insoluble (Hamilton, 1998; Wang et al., 2021a). The led to corrosion. The two half-reactions below can be adopted to illustrate the EET-MIC of carbon steel caused by D. ferrophilus (Wang et al., 2021a):

4Fe → 4Fe²⁺ + 8e⁻ (E^o = − 447 mV_SHE) (extracellular) (3)

SO₄²⁻ + 9H⁺ + 8e⁻ → HS⁻ + 4H₂O (E^o´ = −217 mV_SHE) (intracellular) (4)

The cell potential (∆E^o´) at pH 7 (indicated by the apostrophe) of the combined reaction consisting of Reactions (3) and (4) is + 230 mV, which is a positive value indicating a thermodynamically favorable corrosion process with energy release that benefits SRB metabolism (Wang et al., 2020).

SEM images of the X80 square coupons with 100 ppm THPS treatment and no treatment after the 7-d incubation are shown in Fig. 7. In the presence of 100 ppm THPS, a corrosion product film with cracks is observed on the surface of the coupon in Fig. 7(a). The cracks could be due to the dehydration procedure in the preparation of the coupon for biofilm imaging under SEM. In comparison, without treatment, the coupon was completely covered with biofilm biomass on top (Fig. 7(b)).

Figure 8 shows the XPS spectra of Fe 2p on the surface of the X80 square coupons after the 7-d SRB incubation. The relative contents of the key components and binding energies of the corrosion products are listed in Table 3. In the presence of 100 ppm THPS, there were three peaks located at 710.3 eV, 711.2 eV, and 709.8 eV, corresponding to FeO, Fe₂O₃, and Fe₃O₄, respectively (Wang et al., 2023; Yang et al., 2020). These peaks were also observed on the coupon with no treatment. The coupon with no treatment had additional FeS and FeS₂ peaks located at 712.6 eV and 707.1 eV, respectively (Wu et al., 2021; Zhao et al., 2007). These additional iron sulfide peaks were typical in SRB MIC of carbon steel (Liang et al., 2014; Shiibashi et al., 2020), because SRB reduced SO₄²⁻ to S²⁻ and HS⁻. Then, S²⁻ and HS⁻ combined with Fe²⁺ released by iron oxidation to form FeS and FeS₂.

Table 3

XPS results showing relative contents of key components in corrosion products of X80 coupon after 7-d immersion in *D. ferrophilus* broth with 100 ppm THPS and without THPS in EASW.
Treatment	Corrosion product	Binding energy (eV)	Atom%
100 ppm THPS	FeO	710.3 ± 0.1	36.3
	Fe₂O₃	711.2 ± 0.1	38.4
	Fe₃O₄	709.8 ± 0.1	26.3
No treatment	FeO	710.3 ± 0.1	21.5
	Fe₂O₃	711.2 ± 0.1	14.8
	Fe₃O₄	709.8 ± 0.1	23.6
	FeS	712.6 ± 0.1	35.4
	FeS₂	707.1 ± 0.2	4.7

As previously shown, the sessile cell count with 100 ppm THPS was much smaller than that on the no-treatment coupon (Fig. 3), resulting in a lower MIC rate (Figs. 5 and 6). This lower MIC rate yielded less corrosion products. Notably, the H₂S concentration in the bottle with 100 ppm THPS treatment was only 8.6% of that with no treatment (Table 2). Therefore, the relative contents of FeS and FeS₂ were too low to be detected with THPS treatment.

Figure 9 exhibits the stress–strain curves for X80 dogbone coupons after the 7-d incubation in abiotic EASW (control) and in D. ferrophilus broth with no treatment and with 100 ppm THPS treatment. The ultimate tensile strength and strain data are listed in Table 4. A high ultimate tensile strain means that a metal is less brittle. Figure 9 shows that the abiotic dogbone in EASW suffered a minor degradation of mechanical properties when compared with pristine dogbone without immersion.

Table 4

Ultimate tensile strength and ultimate tensile strain data from Fig. 9.
Treatment	Ultimate tensile strength (MPa) (and loss)	Ultimate tensile strain (%) (and loss)
Abiotic EASW 100 ppm THPS No treatment	867 (control) 867 (0% loss) 819 (6% loss)	14.0% (control) 13.6% (3% loss) 12.2% (13% loss)

The abiotic dogbone’s ultimate tensile strength of 867 MPa and ultimate tensile strain of 14.0% were used as the basis to calculate the microbial degradation of mechanical properties and the 100 ppm THPS treatment effects. After the 7-d D. ferrophilus incubation with no treatment, the ultimate tensile strength lost 6%. In comparison, with 100 ppm THPS, the X80 dogbone suffered no loss in ultimate tensile strength. Moreover, after the 7-d D. ferrophilus incubation with no treatment and with 100 ppm THPS treatment, the ultimate tensile strain lost 13% and 3%, respectively. This suggests that the MIC treatment mitigated the microbial degradation of X80 mechanical properties by cutting down the losses in ultimate tensile strength and ultimate tensile strain. Therefore, to mitigate the degradation of mechanical properties caused by MIC, MIC itself must be treated Fig. 10 graphically summarizes the main results presented in this study.

In this study, the significant weight loss, pitting and degradation of mechanical properties in the 7-d lab experiment were made possible by the use of a super-corrosive SRB again X80 carbon steel. The mitigation of D. ferrophilus biofilms on the X80 square coupons with 100 ppm THPS resulted in approximately 3-log reduction in sessile cells compared to the no-treatment control. Because fewer sessile cells were present on the treated X80 square coupons, fewer electrons were harvested by the sessile cells from X80, leading to a much lower weight loss of 1.1 mg cm^− 2 (vs. 19.5 mg cm^− 2 for the control). The maximum pit depth dropped from 13.5 µm to 2.2 µm to practically undetectable. As a consequence, the 100 ppm THPS treatment reduced the losses of ultimate tensile strength and ultimate tensile strain. The data in this work convincingly proved the hypothesis that mitigating biofilm and MIC will reduce the microbial degradation of a carbon steel’s mechanical properties.

Acknowledgements

Chinese Society for Corrosion and Protection (CSCP) and Corrosion & Protection Center, University of Science & Technology Beijing financially supported this project. W. Dou received financial support from Institute of Marine Science and Technology, Shandong University, China.

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No competing interests reported.

Mitigation of microbial degradation of X80 carbon steel mechanical properties using a green biocide

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Abstract

Figures

Introduction

Materials and methods

Results and discussion

Biofilm imaging, cell counts, and gas measurements

Weight loss, pit depth, SEM, and XPS analyses

Conclusion

Declarations

Acknowledgements

References

Additional Declarations

Status:

Journal Publication

Version 1