Effect of yeast extract on microbiologically influenced corrosion of X70 pipeline steel by SRB

doi:10.21203/rs.3.rs-2452348/v1

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Effect of yeast extract on microbiologically influenced corrosion of X70 pipeline steel by SRB

https://doi.org/10.21203/rs.3.rs-2452348/v1

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Microbiologically influenced corrosion (MIC) is a complex process involving interactions between microorganisms, materials and surrounding environment. Yeast extract (YE) is a routinely added nutritional ingredient in many kinds of microbial culture media. However, the effect of YE on MIC was not clear. In this work, the corrosion behaviors of X70 pipeline steel in the sulfate reducing bacterial (SRB) Desulfovibrio bizertensis SY-1 inoculated media with or without YE were studied. The results showed that the weight loss of coupons in sterile media increased twofold after removing YE from culture media (P = 0.04). While in the SRB culture medium, removing YE decreased the planktonic bacteria number, but significantly promoted the attachment of bacteria on steel surfaces. Besides, the corrosion rate of steel in SRB media without YE was four times higher than with YE (P = 0.003). MIC was not determined by the planktonic SRB cells but the attached biofilm. The results identified effect of YE on D. bizertensis SY-1 growth and confirmed the inhibition role of YE in MIC induced by SRB.

Earth and environmental sciences/Ocean sciences/Marine chemistry

Biological sciences/Microbiology/Biofilms

Microbiologically influenced corrosion

Sulfate-reducing bacterial

Yeast extract

Extracellular electron transfer

Corrosion Inhibition

Microorganisms can get involved into the corrosion process of metallic materials directly or indirectly via microbial metabolic activities^1,2, which is called as microbiologically influenced corrosion (MIC). Some studies indicated that 70% of microbial corrosion was caused by sulfate-reducing bacterial (SRB)³. SRB are a class of prokaryotic microorganisms that are capable of obtaining energy by reducing sulfate, sulfite or thiosulfate to H₂S as the final electron acceptor⁴. SRB are widely spread in the various environments, like seawater⁵, soil⁶, oil⁷ and gas transportation pipelines^7,8, and they can cause corrosion of almost all metals, like iron⁹, stainless steel¹⁰, copper¹¹, aluminum and other alloys^12–14.

Currently, most studies on MIC rely on single or mixed bacteria in defined culture media under laboratory conditions¹⁵. Thus, the choice of proper culture medium was very important as the environment factors in aqueous solution were particularly important in the corrosion studies^{9, 16–18}. The commonly used culture media used for SRB were shown in Table S1 and mainly include Postgate's B (PGB) medium¹⁹, Postgate's C (PGC) medium¹⁹, ATCC1249 medium²⁰, etc. In these nutrient rich containing culture media, SRB used lactate as the electron donor and sulfate as the electron acceptor²¹. In fact, there were significant differences in the role of different nutrient components for the metabolic activity of SRB cells. For example, the addition of phosphate in the medium promoted the formation of the protective film of the coupon and slowed down its corrosion rate^22,23. For SRB with specific functions, researchers also designed different culture medium according to the particular environment. For example, Dinh et al.⁹ isolated and cultured new SRB (strains IS4 and IS5) from the marine environment by using iron as the only electron donor, which could capable of directly utilizing iron electrons.

As a routinely nutritional ingredient added in microbiological culture media, yeast extract (YE) could be found in many kinds of culture media lists^24–26. YE is a complicated natural resource which is generally obtained from brewer's or baker's yeast at a temperature of 50°C²⁷. Carbs, amino acids, peptides, vitamins, and trace elements are all involved in YE²⁷. Lately, YE was also utilized as an additional ingredient in serum-free medium for the growth of mammalian cells and the generation of immunoglobulin²⁸. It was found that YE significantly increased the biomass of most bacteria and their growth rate. For example, the addition of YE to the medium significantly promoted the growth of Lactobacillus plantarum, but there could be a 50% difference in bacterial growth with YE produced by different manufacturing processes²⁹. Similarly, Sulfobacillus thermosulfidooxidans cell growth and its iron oxidation process were both increased with the enhanced concentration of YE³⁰. Besides, certain amino acids in YE media (Asp, Val, Glu, and Try) promoted Escherichia coli growth³¹, while other amino acids (Ser, Ile, et al.) had no influence on E. coli cell growth. Meanwhile, the presence of YE significantly promoted the metabolic processes of some kinds of bacteria^32,33. For example, when 0.05 g/L YE was added, the degradation of C8 ~ C14 hydrocarbons by Acinetobacter baumannii was doubled³⁴. YE, as an effective nitrogen source, stimulated the growth of Rhodobacter sphaeroides cells in mineral springs and improved H₂ production³⁵. The removal of YE resulted in 33% decrease in the ability of Clostridium kluyveri to degrade fatty acids to hexanoic acid during secondary fermentation³⁶. However, in contrast, Saksinchai et al.²⁸ found that YE restricted the cell growth, by-product formation and sporulation of Bacillus thuringiensissubsp. Kurosaki. Therefore, there are large differences in the metabolic processes of YE on the growth of different bacteria.

Recently, another popular culture media, namely, Zellkulturen (DSMZ) 195c medium, was used in SRB culturation and MIC study, especially in the study focused on extracellular electron transfer (EET) between microbes and electrodes³⁷. Notably, the biggest difference between DSMZ 195 culture media and other media is whether or not the culture medium contains YE. One of the main causes for removing YE from culture media was its interference in corrosion behavior. The redox compounds may participate in the electrochemical reactions, producing a significant current signal and affecting the analysis of EET processes in MIC process³⁸. However, the macromoleculars contained in YE may also absorb on the metal surface and acted as an inhibitor against further corrosion^39,40.

Addition of YE in aqueous solution has a complex effect on MIC. On one hand, its stimulation in bacteria growth may cause severe corrosion and on the other hand, its absorption on steel surfaces may also reduce the corrosion rate. Thus, the effect of YE on the growth process of SRB and the resulting corrosion is still unclear. Finding out the effects of YE on bacterial metabolic activity and corrosion process helps the further design and optimization of medium components. In the current work, the corrosion behaviors of X70 pipeline steel in SRB culture media with or without YE in anaerobic culture media were investigated trying to find the specific effect of YE on MIC induced by SRB.

Growth curve of SRB

Figure 1a showed the variation of planktonic cell counts in SRB culture media with or without YE. In the culture medium with YE, SRB growth entered the logarithmic growth phase and multiplied rapidly during the first 9 days, with the cell count reaching a maximum of 6.62 × 10⁸ cells mL^− 1 on the 9th day. After that, the bacteria cells decreased rapidly in the next 2 days, and then stabilized around 2.28 × 10⁸ cells mL^− 1. This decline could be explained by the quick nutrient consumption in the culture medium, which caused a decrease in cell concentration.

However, for the culture medium without YE, there was a 5-day lag period once after inoculation, and after that the SRB cells started increase at a much slower rate than that in PGC media with YE. The planktonic cell concentration reached a maximum value of 2.87 × 10⁸ cells mL^− 1 on the 10th day and then stabled. The planktonic cell concentration in culture medium with YE or without YE were similar after inoculation for 10 days. There was a significant promotion in SRB planktonic cells growth in PGC culture media with YE addition during the first 10 days.

Figure 1b showed the variation of pH in different culture media throughout the SRB culture phase. The initial pH (around 6.50) of the culture media with YE was much lower than that without YE (around 6.86), and then the pH increased and fluctuated during the rapid cell growth phase. While the pH in the culture media without YE increased slightly during the growth phase.

Surface analysis

After immersing in different media for 15 days, the X70 pipeline steel coupons were taken out for SEM observations. In the sterile culture medium without YE (Fig. 2e and f), the dense film of corrosion products formed on the coupon surface presented lamellar and irregular spherical morphologies. Relatively, the corrosion morphologies of coupons in the sterile culture medium without YE (Fig. 2g and h) were similar to that in the YE medium, excepted that a large number of cracks and a large aggregation of irregular inorganic corrosion products were observed in YE-removed system.

In SRB-inoculated medium, both with and without YE, many adhering cells were observed on the coupons' surface. When YE was taken out of the culture media, nonetheless, a substantially greater number of sessile cells were seen on the coupon surface. In the biotic media with YE (Fig. 2a and b), some amorphous biofilm and inorganic corrosion products were found. SRB cells produced a large amount of extracellular polymer (EPS), which combined the attached cells and inorganic corrosion products together and formed a dense biofilm covering the coupon completely. Within the biofilm, SRB cells were found firmly attached to the coupon surface (Fig. 2d).

XPS analysis of corrosion products

The XPS spectra of O 1s, Fe 2p and S 2p of the X70 coupons after 15 days' immersion in the various growth mediums was shown in Fig. 3. The composition of the corrosion products in SRB media with and without YE were similar. The core level Fe 2p spectrum was identified into four peaks at E_b of 708.08, 710.60, 713.60, and 722.85 eV respectively caused by FeS₂, Fe₃O₄, FeS, and FeOOH⁴¹. It was found that there were four distinct peaks in the core level O 1s spectrum. The Fe₃O₄ and FeOOH corresponded to 530.42 and 531.39 eV, respectively. The inorganic SO₄²⁻ was detected at 532.09 eV, and the organic C-O compounds were also detected at 536.19 eV, which could be attributed to the EPS produced by SRB metabolism⁴². In the S 2p spectrum, peaks at E_b 161.01, 162.31 and 168.64 eV were attributed to FeS₂, FeS and SO₄²⁻, respectively⁴³.

In the sterile culture medium, element S was hardly detected and the coupons differed considerably in iron oxide composition. In the abiotic culture with YE, the Fe 2p spectrum consisted of dominant peaks of Fe₃O₄ (710.31 eV), FePO₄ (712.43 eV) and FeOOH (724.37 eV). While for the coupons in abiotic medium without YE, only Fe 2p spectrum of Fe₃O₄ and FeOOH were detected, but not FePO₄ peak was found.

Corrosion rate and pitting depth measurements

To more quantitatively determine the effect of YE on the corrosion of X70 pipeline steel induced by D. bizertensis SY-1, the weight loss of coupons under different conditions were tested after 15 days of immersion and the results were shown in Fig. 4. The corrosion rate of coupons in the SRB culture media was significantly higher than that in sterile medium. The corrosion rate of coupons in the SRB culture medium without YE (100.17 ± 5.63 mm y^− 1) was four times higher than that in SRB-inoculated medium with YE (25.59 ± 4.67 mm y^− 1), and there was a very significant difference (p = 0.0034) between the corrosion rate of X70 pipeline steel in SRB media with or without YE. While for the coupons in abiotic medium with or without YE, there was significant difference (p = 0.04) between corrosion rate. For the coupons in sterile media without YE, the corrosion rate (6.94 ± 2.21 mm y^− 1) was twice as high as that in the presence of YE (3.33 ± 2.17 mm y^− 1). The presence of YE significantly inhibited the corrosion of X70 pipeline steel in both sterile and SRB media.

After removing the corrosion products, obvious pitting was clearly observed on the coupons in all the systems (Fig. 5). The maximum depth of the pits formed by the SRB biofilm in the absence of YE was 22.483 µm, which was almost twice as deep as that of the pits formed in the presence of YE. Similarly, the maximum depth of pits formed under the sterile medium without the presence of YE was 7.737 µm, which was 1.6 times greater than that formed in the presence of YE. These results suggested that local corrosion was intensified in the absence of YE. The adhesion of YE on the coupon surface resulted in the formation of a dense film of corrosion products⁴⁴, which could prevent further dissolution of iron and provide protection to the coupon. The presence of SRB significantly promoted the process of pitting, which was in consistent with previous studies that attached SRB cells could act as biocathode in EET and cause severe pitting by direct electron transfer (DET)^45,46.

Electrochemical test results

The coupons were electrochemically tested every other day. Figure 6 showed the changes of E_ocp with respect to the immersion time under different conditions. The results showed that the E_ocp shifted positively during the first 3 days in the SRB culture media with or without YE, and then gradually stabilized. Similarly, the E_ocp of coupons in the sterile medium with YE increased during the first 7 days and then remained stable at -0.70 V vs. SCE. While for coupons in the sterile medium without YE, the E_ocp fluctuated during the first 5 days, and then remained around − 0.72 V vs. SCE from days 7 to 15. The E_ocp of the coupons in SRB medium was more positive than that in the sterile medium, which could be related to the formation of biofilm. Besides, the addition of YE lead to the positive shift of E_ocp in both sterile and SRB medium.

LPR is a popular electrochemical technique for acquiring MIC data, including corrosion rate and polarization resistance (R_p)⁴⁷. According to Fig. 7, the higher R_p during the first three days for the coupons immersed in the sterile medium suggested a reduction in the corrosion rate, which could be attributable to the deposition of corrosion products on the coupon surface at the beginning of immersion⁴⁸. After 3 days of immersion, the value of R_p in the sterile culture medium with YE increased sharply, indicating that corrosion was significantly inhibited. This could be the result of YE adhering to the coupon surface and forming a thick deposit of a corrosion product made of FePO₄ (Fig. 2f). The R_p of coupons in both SRB media with or without YE showed a continuous downward trend during the first 5 days, indicating an increased corrosion rate. Subsequently, the constant R_p was maintained, with a higher R_p value observed in SRB culture media with YE addition than that without YE. These results were in consistent with the SEM morphology and weight loss data, which confirmed that the corrosion rate of X70 pipeline steel was drastically increased in the SRB and YE-free medium.

Figure 8 displayed the EIS results for X70 pipeline steel in various culture medium. The Nyquist plot radius for the coupons in the sterile media containing YE declined from day 1 to day 5, and then increased significantly. Meanwhile, the phase angle peak in the Bode graphs shifted from low to high frequency after 5 days. The protective effect of the corrosion product layer was often given as the reason for these phenomena⁴⁸. According to the results of SEM (Fig. 2f), the formation of a thick corrosion product layer was found on the coupon surface immersed in the sterile medium with YE, and this layer was mostly composed by FePO₄, Fe₃O₄ and FeOOH (Fig. 3), which were reported to inhibit corrosion by forming deposits⁴². Conversely, for the coupons in the sterile media without YE, the radius of the Nyquist plot consistently decreased. In the SRB medium without YE, the radius of the Nyquist curve decreased within 15 days, indicating that the attachment of SRB to the coupon surface formed the biofilm significantly promoted corrosion. However, for the coupons in SRB media with YE, the slightly increased radius of the Nyquist diagram could be caused by YE adsorption and SRB metabolic activities. Furthermore, for the whole time period, the Nyquist plot radius in the presence of YE was greater than that in the absence of YE, indicating that MIC was inhibited in the presence of YE.

Supplementary Fig. 1 displayed the equivalent circuit that was determined to most accurately represent the impedance diagram. ZSimpWin was used to fit the curves, and the chi-square (χ²) value was found to be less than 0.001. As an alternative to capacitance, the constant phase element (CPE or Q) was taken into account when modeling distributed capacitance. The impedance of Q was calculated from the following Eq. 1³:

Z _Q=Y₀⁻¹(jω)⁻ⁿ (1)

where Y₀ was the CPE intensity and n was an empirical exponent reflecting the surface heterogeneity. The j was the irrational unit, and ω was the angular frequency (rad/s)⁴⁸. The suggested models defined the solution resistance as R_s. The capacitance of the film that included biofilm and corrosion products was defined by Q_f. R_f indicated for the biofilm and corrosion product-containing film's resistance. The double layer capacitance and the charge transfer resistance defined by Q_dl and R_ct, respectively.

Figure 9 displayed the time-dependent shifts in R_ct and R_f. These two values were significantly greater in the YE-containing sterile medium as a result of immersion time, and they were much higher than that in the sterile media without YE. For the coupons in SRB media, the lower R_ct observed in SRB media compared with that in sterile media indicated the accelerated corrosion by SRB. The R_f in the SRB medium containing YE got its maximum value on the 15th day, which was 3 times higher than that in the SRB medium without YE. In the SRB medium without YE, the R_ct decreased constantly with time, indicating an increasing corrosion rate over the whole immersion time. Besides, the lower R_ct of coupons in SRB medium without YE than those in SRB media with YE could be attributed to the dense SRB biofilm formed on steel surface when YE was removed³. Those sessile SRB cells acted as biocathode in EET and played an important role in accelerating MIC³. Besides, the coupons in the SRB medium without YE showed a much smaller R_f values than that in SRB medium with YE, indicating that SRB metabolism produced a large amount of EPS and formed a highly conductive FeS corrosion product layer on the coupon surface⁴⁹.

After 15 days immersion in the SRB and sterile culture media with and without YE, the coupons' potentiodynamic polarization curves were shown in Fig. 10. The findings demonstrated that the coupons in the SRB medium had a greater corrosion current density (I_corr) compared to those in the sterile medium. For the coupons in different environment, the highest I_corr was found in the SRB medium without YE (28.73 ± 1.72 nA cm^− 2), which was 14 times higher than that in SRB medium with YE (2.47 ± 0.39 nA cm^− 2). The lowest I_corr was found in sterile media with YE (1.34 ± 0.98 nA cm^− 2), followed by that in the sterile media without YE (2.47 ± 0.39 nA cm^− 2). Consistent with LPR and EIS findings, the results provided more evidence that YE inhibited the corrosion induced by SRB. These electrochemical findings, as well as the weight loss findings and SEM observations, corroborated the accelerated impact of SRB on X70 pipeline steel corrosion and the inhibiting effect of YE on MIC.

Table 1

**Tafel parameters of the coupons exposed to culture medium with and without YE** (b_a: Tafel slope of the anodic curve; b_c: Tafel slope of the cathodic curve).
Condition	SRB with YE	SRB without YE	Sterile with YE	Sterile without YE
I_corr (nA cm^− 2)	2.47 ± 0.39	28.73 ± 1.72	1.34 ± 0.98	2.47 ± 0.39
E_corr (V vs SCE)	-0.79 ± 0.01	-0.92 ± 0.01	-0.79 ± 0.01	-0.76 ± 0.01
b_a (V dec^− 1)	0.30 ± 0.05	0.47 ± 0.05	0.18 ± 0.01	0.07 ± 0.01
b_c (V dec^− 1)	0.12 ± 0.01	0.03 ± 0.001	0.15 ± 0.02	0.16 ± 0.01

YE acts as a natural corrosion inhibitor in the corrosion of X70 pipeline steel

YE contains some redox mediators, like vitamins B, riboflavin and nicotinamide, which were inclined to adsorbed on metal surface and chelate metal ions²⁷. Paul et al. ³⁹ found that YE contains benzene ring organic compounds such as amines, diamines and pyridines, which had a strong corrosion inhibition effect on steel in the aqueous environment. These natural corrosion inhibitors had a maximum adsorption rate of 70–90%⁵⁰. Chidiebere et al.⁵⁰ found that low concentrations of riboflavin had a corrosion inhibition effect on mild steel under acidic conditions. Chang et al.⁴⁴ also found that exogenous riboflavin acted as the electron transfer carrier to form a dense phosphorylated layer on the surface of pure iron, thereby inhibiting corrosion.

In the sterile medium without YE, it could be clearly seen from the SEM morphology (Fig. 2f) that a thick corrosion product layer was formed, greatly reducing the corrosion rate and local pitting corrosion of X70 pipeline steel. In addition, electrochemical data also showed that R_f and R_ct values were shown to be higher in the YE-free culture media compared to the YE-containing medium.

Sessile SRB cells obtain electrons directly from Fe(0)

Based on bioenergetics, SRB cells preferred to use soluble nutrient as carbon sources in metabolic activities when sufficient carbon sources were supplied⁵¹. However, the SRB cells have to obtain electrons and energies from iron or polarized electrodes^52,53 in energy-limited environments^54,55, thus causing severe metal corrosion by obtaining electrons from iron⁵⁶.

When bacteria cells were in a “starved” state (no or only a small amount of carbon source provided), it usually leaded to more severe corrosion than when the carbon source was abundant, which indicated that SRB biofilms corrode metals via obtaining energy from iron to sustain its life activities^57–60. Previous studies also demonstrated that Desulfovibrio could directly obtain electrons from Fe(0)^53,61.

In this study, more sessile cells and less planktonic SRB cells were found in SRB media without YE compared with that in SRB media with YE. Meanwhile, higher corrosion rate and deeper corrosion pits were found in SRB media without YE. The results demonstrated that the X70 steel coupon corrosion was accelerated by SRB and the MIC was mainly determined by the sessile SRB cells but not the planktonic SRB cells.

Effect of YE on X70 pipeline steel corrosion induced by SRB

The nitrogenous compounds and carbohydrates contained in YE can stimulate the growth of some microorganisms⁶². The removal of YE from the culture media decreased the rate of SRB development because YE was a possible "redox agent" in the medium²⁷. However, not all SRB strains responded well to YE in terms of growth. According to research by F. Widdel et al.¹⁹, certain gram-negative SRB strains were not encouraged by complex nutrients, and YE even seemed to be a growth inhibitor for this particular bacterial group.

In PGC culture media, lactate and YE were added as carbon sources for microbial metabolism. The addition of YE mainly promoted the initial growth of planktonic SRB cells. The concentration of planktonic cells reached the highest value on the 9th day, and the number was twice as high as they were in the medium without YE. After long-term cultivation, the concentration of planktonic cells was almost the same in SRB medium with or without YE.

In contrast, YE could have the opposite impact on the sessile cells that attached to the surface of the coupon. Steel coupons would rapidly absorb YE from the surrounding media once they were immersed in it⁵⁰. Once the YE was removed from the, the SRB cells were inclined to form biofilm on steel surface and obtain electrons from iron for survival. Therefore, the corrosion rate induced by SRB was more serious without YE, which was 4 times higher than that in the medium with YE (p < 0.01). After 15 days’ immersion in sterile media, the maximum pitting depth in the absence of YE was twice as that in the presence of YE, and the coupon showed no visible pitting in the YE-containing system.

The SEM morphology observation also found that in the culture medium without YE, a complete and dense biofilm and abundant EPS was clearly showed on the coupon surfaces, which might facilitate the direct electron acquisition of attached SRB cells from steel. However, only a few bacterial cells were observed adhered onto the coupon with YE addition. The electrochemical data also showed that the R_ct values of the coupon in the SRB media with YE were significantly higher than that without YE, indicating a favored EET process between biofilm and steel surface.

Synergistic effect of YE removal and D. bizertensis SY-1 on the corrosion of X70 pipeline steel

The corrosion of X70 pipeline steel in the SRB inoculated medium was influenced by metal material properties, microbial metabolic activity, and environmental variables. Thus, the synergistic impact of YE removal and D. bizertensis SY-1 on the corrosion of X70 pipeline steel could be characterized as:

$${WL}_{(YE-removal, SRB)}={WL}_{\left(PGC\right)}+\varDelta {WL}_{(YE-removal)}+\varDelta {WL}_{\left(SRB\right)}+\varDelta {WL}_{s(YE-removal, SRB)}$$

Where, WL_(PGC) presented the corrosion rate of the coupon in the sterile PGC medium and it reflected the nature of the steel in PGC solution. While, ΔWL_{(YE−removal)} and ΔWL_(SRB) presented the change in corrosion rate due to YE removal and SRB inoculation. The change in corrosion rate caused by the synergistic role of YE removal and SRB was presented by ΔWL_{(YE−removal,SRB)}. Taking these corrosion rate data into calculation, the values ofΔWL_{(YE−removal)}, ΔWL_(SRB) and ΔWL_{(YE−removal,SRB)} were 3.62, 22.26 and 74.58.

Results demonstrated that both the ΔWL_{(YE−removal)} and ΔWL_(SRB) had positive roles in accelerating corrosion. Further, the combined effect of YE removal and SRB inoculation was much greater in accelerating corrosion than either factor alone.

YE acted as a natural corrosion inhibitor in the corrosion pipeline steel in the abiotic media. For the coupons in the sterile media, the inhibition may occur by YE adsorption on metal surface and combination with ions to form a dense corrosion product film. In the SRB media, YE acted as a source of organic carbon and nitrogen in the culture solution (Fig. 11), which significantly promoted the growth of planktonic cells. However, for the attached cells, the SRB cells within the biofilm would use the YE adsorbed on the surface of the coupon as an energy source to promote their own growth and metabolism, thus not directly obtained electrons from iron. Therefore, yeast extract could not be recommended in culture medium while focusing on the EET process between the interface of biofilm and electrode. YE provided the sufficient carbon source for the sessile SRB cells, thereby inhibiting the corrosion caused by cells obtained electrons from iron.

Bacterium, culture medium and materials

Marine isolated SRB strain Desulfovibrio bizertensis SY-1 (NCBI: PRJNA790473) from the rust layers exposed in the South China Sea, was used in this study. D. bizertensis SY-1 was cultivated in PGC medium, and Sinopharm Chemical Reagent Co., Ltd. supplied all of the chemicals utilized (China). In order to keep anaerobic condition, L-cysteine was introduced to the sterile medium with 100 ppm (w/w) as an oxygen scavenger using a disposable 0.22 µm Stericup filter (Millipore, Bedford, MA). In order to study the effect of YE on the growth of D. bizertensis SY-1 and corrosion of the coupon, cultivation and experiments were conducted in full PGC medium or YE-removed PGC medium. For the YE-removed PGC medium, the other ingredients in YE-removed PGC medium are same as the full PGC medium, except for the absence of YE.

In this work, X70 pipeline steel with the following elemental composition (wt %) was used: 0.13 C, 0.29 Si, 1.65 Mn, 0.01 S, 0.02 P, 0.03 V, and balance Fe. The P600, P1000, and P2000 grit SiC papers were used to grind the X70 pipeline steel coupons, which were then washed in pure ethanol and dried with nitrogen. Before the testing, all coupons were sterilized for 30 minutes under the UV light.

Immersion tests

The immersion experiments were conducted using X70 pipeline steel coupons that were 10 × 10 × 3 mm in size. The 100 mL anaerobic vials were used for immersion tests. One coupon was fixed in each vial with the 50 mL sterile culture media. Each vial was inoculated anaerobically with 0.5 mL (1% v/v) 5-day-old bacterial culture, and then cultured at 30°C for 15 days.

The number of SRB cells in the culture medium was counted by the direct counting method using a microscope. (Olympus, IX73). The pH meter (METTLER TOLEDO, S220-B) was employed to monitor the culture medium.

Surface characterization

The scanning electron microscopy (SEM, FEI Quanta 250) was used to examine the structure of the corrosion layer. After immersion for 15 days, the coupons were fixed in a 2.5% glutaraldehyde solution for 2 hours. Then, gradient ethanol concentrations of 25%, 50%, 60%, 70%, 80%, 90%, and 100% were used to dehydrate the coupons for a total of 10 minutes. The coupon surface was plated with Au for conductivity before SEM observation.

The chemical makeup of the corrosion products was identified with the X-ray photoelectron spectroscopy (XPS, ESCALAB 250XI, Thermo Fisher). A 50.0 eV analyzer pass energy was used to power the XPS photoelectrons, which were generated by an Al Ka (1486.6 eV) X-ray source. The binding energies (E_b) at 284.8 eV were calibrated using the surface carbon contamination.

Weight loss experiments

The X70 pipeline steel coupons were withdrawn from the various media after 15 days immersion, and its corrosion products were cleaned in accordance with ISO 8407:2009, IDT⁶³. The weight loss experiments were got via the electronic balance (QUINTIXI124-1CN). Three duplicate samples for each system were used. The formula that was used to determine the corrosion rate of coupons was as follows:

$$Corrosion rate (mm/y)=\frac{8.76\times {10}^{4}\times weight loss\left(g\right)}{density\left(g\bullet {cm}^{-3}\right) \times area\left({cm}^{2}\right) \times time\left(h\right)}$$

Electrochemical measurements

The electrochemical measurements were carried out using a conventional three-electrode setup with sterilized or SRB inoculated culture medium⁶⁴. The working electrode was made up of steel coupons from the X70 pipeline measuring 10 × 10 × 10 mm. The reference electrode was a saturated calomel electrode (SCE), and the counter electrode was a platinum plate. An electrochemical workstation carried out each of the electrochemical experiments (Gamry, Reference 3000). After the open circuit potentials (E_ocp) stabilized, electrochemical impedance spectroscopy (EIS) experiments were carried out with a sinusoidal perturbation of 10 mV in the frequency range of 10⁵ to 10^− 2 Hz. After the 15-day immersion, polarization curves were obtained with a scanning rate of 0.167 mV/s from − 250 mV to + 250 mV vs E_ocp⁶³. These electrochemical tests were carried out on three duplicate samples.

DATA AVAILABILITY

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

ACKNOWLEDGEMENTS

This work was supported financiaslly by the National Natural Science Foundation of China (No. 41806090, 42076044), and the Key Research Program of Frontier Sciences, CAS (ZDBS-LY-DQC025).

AUTHOR CONTRIBUTIONS

Y.P.: Methodology, Software, Investigation, Writing—Original Draft; Y.Z., R.Z., X.Z.: Resources, Visualization, Investigation; F.G., J.D.: Writing—Review & Editing, Conceptualization, Project administration, Funding acquisition; B.H.: Supervision

COMPETING INTERESTS

The authors declare no competing interests.

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Effect of yeast extract on microbiologically influenced corrosion of X70 pipeline steel by SRB

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Abstract

Figures

Introduction

Results

Growth curve of SRB

Surface analysis

XPS analysis of corrosion products

Corrosion rate and pitting depth measurements

Electrochemical test results

Discussion

YE acts as a natural corrosion inhibitor in the corrosion of X70 pipeline steel

Sessile SRB cells obtain electrons directly from Fe(0)

Effect of YE on X70 pipeline steel corrosion induced by SRB

Methods

Bacterium, culture medium and materials

Immersion tests

Surface characterization

Weight loss experiments

Electrochemical measurements

Declarations

References

Additional Declarations

Supplementary Files

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