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Microbial corrosion (MIC) is a serious problem in many industries as it can cause huge economic losses.2707 super duplex stainless steel (2707 HDSS) has been used in marine environments due to its excellent chemical resistance.However, its resistance to MIC has not been experimentally demonstrated.In this study, the MIC behavior of 2707 HDSS caused by the marine aerobic bacterium Pseudomonas aeruginosa was investigated.Electrochemical analysis showed that in the presence of Pseudomonas aeruginosa biofilm in 2216E medium, there was a positive change in corrosion potential and an increase in corrosion current density.X-ray photoelectron spectroscopy (XPS) analysis showed a decrease in Cr content on the surface of the specimen beneath the biofilm.Imaging analysis of the pits showed that the P. aeruginosa biofilm produced a maximum pit depth of 0.69 μm during 14 days of incubation.Although this is small, it indicates that 2707 HDSS is not fully immune to the MIC of P. aeruginosa biofilms.
Duplex stainless steels (DSS) are widely used in various industries for their ideal combination of excellent mechanical properties and corrosion resistance1,2.However, localized pitting still occurs and it affects the integrity of this steel3,4.DSS is not resistant to microbial corrosion (MIC)5,6.Despite the wide range of applications of DSS, there are still environments where the corrosion resistance of DSS is not sufficient for long-term use.This means more expensive materials with higher corrosion resistance are required.Jeon et al7 found that even super duplex stainless steels (SDSS) have some limitations in terms of corrosion resistance.Therefore, super duplex stainless steels (HDSS) with higher corrosion resistance are required in some applications.This led to the development of highly alloyed HDSS.
The corrosion resistance of DSS depends on the ratio of alpha and gamma phases and the Cr, Mo and W depleted regions 8, 9, 10 adjacent to the second phase.HDSS contains high content of Cr, Mo and N11, so it has excellent corrosion resistance and a high value (45-50) Pitting Resistance Equivalent Number (PREN), determined by wt.% Cr + 3.3 (wt.% Mo + 0.5 wt% W) + 16 wt% N12.Its excellent corrosion resistance relies on a balanced composition containing approximately 50% ferrite (α) and 50% austenite (γ) phases, HDSS has better mechanical properties and higher resistance than conventional DSS13. Chloride corrosion properties.The improved corrosion resistance expands the use of HDSS in more corrosive chloride environments, such as marine environments.
MICs are a major problem in many industries such as oil and gas and water utilities14.MIC accounts for 20% of all corrosion damage15.MIC is bioelectrochemical corrosion that can be observed in many environments.Biofilms that form on metal surfaces alter the electrochemical conditions, thereby affecting the corrosion process.It is widely believed that MIC corrosion is caused by biofilms.Electrogenic microorganisms corrode metals to obtain sustaining energy to survive17.Recent MIC studies have shown that EET (extracellular electron transfer) is the rate-limiting factor in MIC induced by electrogenic microorganisms.Zhang et al. 18 demonstrated that electron mediators accelerate electron transfer between Desulfovibrio sessificans cells and 304 stainless steel, leading to more severe MIC attack.Enning et al. 19 and Venzlaff et al. 20 showed that corrosive sulfate-reducing bacteria (SRB) biofilms can directly absorb electrons from metal substrates, resulting in severe pitting corrosion.
DSS is known to be susceptible to MIC in environments containing SRB, iron-reducing bacteria (IRB), etc. 21 .These bacteria cause localized pitting on DSS surfaces under biofilms22,23.Unlike DSS, the MIC of HDSS24 is poorly known.
Pseudomonas aeruginosa is a gram-negative motile rod-shaped bacterium that is widely distributed in nature25.Pseudomonas aeruginosa is also a major microbial group in the marine environment, causing MIC to steel.Pseudomonas is closely involved in corrosion processes and is recognized as a pioneer colonizer during biofilm formation.Mahat et al. 28 and Yuan et al. 29 demonstrated that Pseudomonas aeruginosa has a tendency to increase the corrosion rate of mild steel and alloys in aqueous environments.
The main objective of this work was to investigate the MIC properties of 2707 HDSS caused by the marine aerobic bacterium Pseudomonas aeruginosa using electrochemical methods, surface analytical techniques and corrosion product analysis.Electrochemical studies including Open Circuit Potential (OCP), Linear Polarization Resistance (LPR), Electrochemical Impedance Spectroscopy (EIS), and Potential Dynamic Polarization were performed to study the MIC behavior of 2707 HDSS.Energy dispersive spectrometer (EDS) analysis was performed to find chemical elements on the corroded surface.In addition, X-ray photoelectron spectroscopy (XPS) analysis was used to determine the stability of oxide film passivation under the influence of a marine environment containing Pseudomonas aeruginosa.The pit depth was measured under a confocal laser scanning microscope (CLSM).
Table 1 lists the chemical composition of 2707 HDSS.Table 2 shows that 2707 HDSS has excellent mechanical properties with a yield strength of 650 MPa.Figure 1 shows the optical microstructure of solution heat treated 2707 HDSS.Elongated bands of austenite and ferrite phases without secondary phases can be seen in the microstructure containing about 50% austenite and 50% ferrite phases.
Figure 2a shows open circuit potential (Eocp) versus exposure time data for 2707 HDSS in abiotic 2216E medium and P. aeruginosa broth for 14 days at 37 °C.It shows that the largest and significant change in Eocp occurs within the first 24 hours.The Eocp values in both cases peaked at -145 mV (vs. SCE) around 16 h and then dropped sharply, reaching -477 mV (vs. SCE) and -236 mV (vs. SCE) for the abiotic sample and P, respectively ). Pseudomonas aeruginosa coupons, respectively.After 24 hours, the Eocp value of 2707 HDSS for P. aeruginosa was relatively stable at -228 mV (vs. SCE), while the corresponding value for non-biological samples was approximately -442 mV (vs. SCE).Eocp in the presence of P. aeruginosa was rather low.
Electrochemical testing of 2707 HDSS specimens in abiotic medium and Pseudomonas aeruginosa broth at 37 °C:
(a) Eocp as a function of exposure time, (b) polarization curves at day 14, (c) Rp as a function of exposure time and (d) icorr as a function of exposure time.
Table 3 lists the electrochemical corrosion parameter values of 2707 HDSS samples exposed to abiotic medium and Pseudomonas aeruginosa inoculated medium for 14 days.The tangents of the anodic and cathodic curves were extrapolated to arrive at the intersections yielding corrosion current density (icorr), corrosion potential (Ecorr) and Tafel slopes (βα and βc) according to standard methods30,31.
As shown in Figure 2b, the upward shift of the P. aeruginosa curve resulted in an increase in Ecorr compared with the abiotic curve.The icorr value, which is proportional to the corrosion rate, increased to 0.328 μA cm-2 in the Pseudomonas aeruginosa sample, four times that of the non-biological sample (0.087 μA cm-2).
LPR is a classic non-destructive electrochemical method for rapid corrosion analysis.It was also used to study MIC32.Figure 2c shows the polarization resistance (Rp) as a function of exposure time.A higher Rp value means less corrosion.Within the first 24 hours, the Rp of 2707 HDSS reached a maximum value of 1955 kΩ cm2 for abiotic samples and 1429 kΩ cm2 for Pseudomonas aeruginosa samples.Figure 2c also shows that the Rp value decreased rapidly after one day and then remained relatively unchanged for the next 13 days.The Rp value of the Pseudomonas aeruginosa sample is about 40 kΩ cm2, which is much lower than the 450 kΩ cm2 value of the non-biological sample.
The icorr value is proportional to the uniform corrosion rate.Its value can be calculated from the following Stern-Geary equation,
Following Zou et al. 33, a typical value of the Tafel slope B in this work was assumed to be 26 mV/dec.Figure 2d shows that the icorr of the non-biological 2707 sample remained relatively stable, while the P. aeruginosa sample fluctuated greatly after the first 24 hours.The icorr values of the P. aeruginosa samples were an order of magnitude higher than the non-biological controls.This trend is consistent with the polarization resistance results.
EIS is another nondestructive technique used to characterize electrochemical reactions at corroded interfaces.Impedance spectra and calculated capacitance values of specimens exposed to abiotic media and Pseudomonas aeruginosa solution, Rb resistance of passive film/biofilm formed on the surface of the specimen, Rct charge transfer resistance, Cdl electric double layer capacitance (EDL ) and QCPE Constant Phase Element (CPE) parameters.These parameters were further analyzed by fitting the data using an equivalent circuit (EEC) model.
Figure 3 shows typical Nyquist plots (a and b) and Bode plots (a’ and b’) of 2707 HDSS samples in abiotic medium and P. aeruginosa broth for different incubation times.The diameter of the Nyquist ring decreases in the presence of Pseudomonas aeruginosa.The Bode plot (Fig. 3b’) shows an increase in the magnitude of the total impedance.Information on the relaxation time constant can be provided by the phase maxima.Figure 4 shows the monolayer (a) and bilayer (b) based physical structures and their corresponding EECs.CPE is introduced into the EEC model.Its admittance and impedance are expressed as follows:
Two physical models and corresponding equivalent circuits for fitting the impedance spectrum of the 2707 HDSS specimen:
where Y0 is the magnitude of the CPE, j is the imaginary number or (-1)1/2, ω is the angular frequency, and n is the CPE power index less than unity35.The inverse of the charge transfer resistance (ie 1/Rct) corresponds to the corrosion rate.Smaller Rct means faster corrosion rate27.After 14 days of incubation, the Rct of the Pseudomonas aeruginosa samples reached 32 kΩ cm2, much smaller than the 489 kΩ cm2 of the non-biological samples (Table 4).
The CLSM images and SEM images in Figure 5 clearly show that the biofilm coverage on the surface of the 2707 HDSS specimen after 7 days is dense.However, after 14 days, the biofilm coverage was sparse and some dead cells appeared.Table 5 shows the biofilm thickness on 2707 HDSS specimens after exposure to P. aeruginosa for 7 and 14 days.The maximum biofilm thickness changed from 23.4 μm after 7 days to 18.9 μm after 14 days.The average biofilm thickness also confirmed this trend.It decreased from 22.2 ± 0.7 μm after 7 days to 17.8 ± 1.0 μm after 14 days.
(a) 3-D CLSM image after 7 days, (b) 3-D CLSM image after 14 days, (c) SEM image after 7 days and (d) SEM image after 14 days.
EDS revealed chemical elements in biofilms and corrosion products on samples exposed to P. aeruginosa for 14 days.Figure 6 shows that the content of C, N, O, and P in biofilms and corrosion products is much higher than that in bare metals, because these elements are associated with biofilms and their metabolites.Microbes only need trace amounts of chromium and iron.High levels of Cr and Fe in the biofilm and corrosion products on the surface of the specimens indicate that the metal matrix has lost elements due to corrosion.
After 14 days, pitting with and without P. aeruginosa was observed in 2216E medium.Before incubation, the specimen surface was smooth and defect-free (Fig. 7a).After incubation and removal of biofilm and corrosion products, the deepest pits on the surface of the specimens were examined under CLSM, as shown in Figure 7b and c.No obvious pits were found on the surface of the non-biological control samples (maximum pit depth 0.02 μm).The maximum pit depth caused by Pseudomonas aeruginosa was 0.52 μm after 7 days and 0.69 μm after 14 days, based on the average maximum pit depth of 3 samples (10 maximum pit depth values were selected for each sample) reached 0.42 ± 0.12 μm and 0.52 ± 0.15 μm, respectively (Table 5).These pit depth values are small but important.
(a) Before exposure, (b) 14 days in abiotic medium and (c) 14 days in Pseudomonas aeruginosa broth.
Figure 8 shows the XPS spectra of different sample surfaces, and the chemical compositions analyzed for each surface are summarized in Table 6.In Table 6, the atomic percentages of Fe and Cr in the presence of P. aeruginosa (samples A and B) were much lower than those of the non-biological control samples (samples C and D).For the P. aeruginosa sample, the Cr 2p core-level spectral curve was fitted to four peak components with binding energy (BE) values of 574.4, 576.6, 578.3 and 586.8 eV, which can be attributed to Cr, Cr2O3, CrO3 and Cr(OH)3, respectively (Fig. 9a and b).For non-biological specimens, the Cr 2p core-level spectrum contains two main peaks for Cr (573.80 eV for BE) and Cr2O3 (575.90 eV for BE) in Fig. 9c and d, respectively.The most striking difference between the abiotic and P. aeruginosa samples was the presence of Cr6+ and a higher relative fraction of Cr(OH)3 (BE of 586.8 eV) beneath the biofilm.
The broad XPS spectra of the surface of the 2707 HDSS specimen in the two media are 7 days and 14 days, respectively.
(a) 7 days of exposure to P. aeruginosa, (b) 14 days of exposure to P. aeruginosa, (c) 7 days in abiotic medium and (d) 14 days in abiotic medium.
HDSS exhibits high levels of corrosion resistance in most environments.Kim et al. 2 reported that UNS S32707 HDSS was defined as a highly alloyed DSS with a PREN of more than 45.The PREN value of the 2707 HDSS specimen in this work was 49.This is due to its high chromium content and high molybdenum and Ni levels, which are beneficial in acidic and high chloride environments.In addition, a well-balanced composition and defect-free microstructure are useful for structural stability and corrosion resistance.However, despite its excellent chemical resistance, the experimental data in this work suggest that 2707 HDSS is not completely immune to the MIC of P. aeruginosa biofilms.
Electrochemical results showed that the corrosion rate of 2707 HDSS in P. aeruginosa broth was significantly increased after 14 days compared to non-biological medium.In Figure 2a, a reduction in Eocp was observed in both abiotic medium and P. aeruginosa broth during the first 24 hours.Afterwards, the biofilm has completed covering the surface of the specimen and the Eocp becomes relatively stable36.However, the level of biological Eocp was much higher than that of non-biological Eocp.There is reason to believe that this difference is due to P. aeruginosa biofilm formation.In Fig. 2d, in the presence of P. aeruginosa, the icorr value of 2707 HDSS reached 0.627 μA cm-2, which was an order of magnitude higher than that of the abiotic control (0.063 μA cm-2), which was consistent with the Rct value measured by EIS.During the first few days, impedance values in P. aeruginosa broth increased due to the attachment of P. aeruginosa cells and the formation of biofilms.However, when the biofilm completely covers the surface of the specimen, the impedance decreases.The protective layer is attacked first due to the formation of biofilms and biofilm metabolites.Therefore, the corrosion resistance decreased over time, and the attachment of P. aeruginosa caused localized corrosion.The trends in abiotic media were different.The corrosion resistance of the non-biological control was much higher than the corresponding value of the samples exposed to P. aeruginosa broth.Furthermore, for abiotic samples, the Rct value of 2707 HDSS reached 489 kΩ cm2 on day 14, which was 15 times the Rct value (32 kΩ cm2) in the presence of P. aeruginosa.Therefore, 2707 HDSS has excellent corrosion resistance in a sterile environment, but is not resistant to MIC attack by P. aeruginosa biofilms.
These results can also be observed from the polarization curves in Fig. 2b.The anodic branching was attributed to Pseudomonas aeruginosa biofilm formation and metal oxidation reactions.At the same time the cathodic reaction is the reduction of oxygen.The presence of P. aeruginosa greatly increased the corrosion current density, approximately an order of magnitude higher than the abiotic control.This indicates that P. aeruginosa biofilm increases localized corrosion of 2707 HDSS.Yuan et al29 found that the corrosion current density of 70/30 Cu-Ni alloy increased under challenge of P. aeruginosa biofilm.This may be due to the biocatalysis of oxygen reduction by Pseudomonas aeruginosa biofilms.This observation may also explain the MIC of 2707 HDSS in this work.Aerobic biofilms may also have less oxygen beneath them.Therefore, the failure to re-passivate the metal surface by oxygen may be a contributing factor to the MIC in this work.
Dickinson et al. 38 suggested that the rates of chemical and electrochemical reactions can be directly affected by the metabolic activity of sessile bacteria on the surface of the specimen and the nature of the corrosion products.As shown in Figure 5 and Table 5, both cell number and biofilm thickness decreased after 14 days.This can be reasonably explained that after 14 days, most of the sessile cells on the surface of 2707 HDSS died due to nutrient depletion in the 2216E medium or the release of toxic metal ions from the 2707 HDSS matrix.This is a limitation of batch experiments.
In this work, the P. aeruginosa biofilm promoted the local depletion of Cr and Fe beneath the biofilm on the 2707 HDSS surface (Fig. 6).In Table 6, the reduction of Fe and Cr in sample D compared to sample C, indicating that dissolved Fe and Cr caused by P. aeruginosa biofilm persisted beyond the first 7 days.The 2216E medium is used to simulate marine environments.It contains 17700 ppm Cl-, which is comparable to that found in natural seawater.The presence of 17700 ppm Cl- was the main reason for the reduction in Cr in the 7- and 14-day abiotic samples analyzed by XPS.Compared to P. aeruginosa samples, the dissolution of Cr in abiotic samples was much less due to the strong Cl− resistance of 2707 HDSS in abiotic environments.Figure 9 shows the presence of Cr6+ in the passivation film.It may be involved in the removal of Cr from steel surfaces by P. aeruginosa biofilms, as suggested by Chen and Clayton.
Due to bacterial growth, the pH values of the medium before and after cultivation were 7.4 and 8.2, respectively.Therefore, below the P. aeruginosa biofilm, organic acid corrosion is unlikely to be a contributing factor to this work due to the relatively high pH in the bulk medium.The pH of the non-biological control medium did not change significantly (from an initial 7.4 to a final 7.5) during the 14-day test period.The increase in pH in the inoculation medium after incubation was due to the metabolic activity of P. aeruginosa and was found to have the same effect on pH in the absence of test strips.
As shown in Figure 7, the maximum pit depth caused by P. aeruginosa biofilm was 0.69 μm, which was much larger than that of the abiotic medium (0.02 μm).This is consistent with the electrochemical data described above.The 0.69 μm pit depth is more than ten times smaller than the 9.5 μm value reported for the 2205 DSS under the same conditions.These data demonstrate that 2707 HDSS exhibits better MIC resistance compared to 2205 DSS.This should come as no surprise, as 2707 HDSS has a higher chromium content, providing longer lasting passivation, due to the balanced phase structure without harmful secondary precipitates, making it harder for P. aeruginosa to depassivate and start points eclipse.
In conclusion, MIC pitting was found on the surface of 2707 HDSS in P. aeruginosa broth compared to negligible pitting in abiotic media.This work shows that 2707 HDSS has better MIC resistance than 2205 DSS, but it is not fully immune to MIC due to P. aeruginosa biofilm.These findings aid in the selection of suitable stainless steels and estimated service life for the marine environment.
The coupon for 2707 HDSS is provided by the School of Metallurgy of Northeastern University (NEU) in Shenyang, China.The elemental composition of 2707 HDSS is shown in Table 1, which was analyzed by the NEU Materials Analysis and Testing Department.All samples were solution treated at 1180 °C for 1 hour.Prior to corrosion testing, coin-shaped 2707 HDSS with a top exposed surface area of 1 cm2 was polished to 2000 grit with silicon carbide paper and further polished with a 0.05 μm Al2O3 powder suspension.The sides and bottom are protected by inert paint.After drying, the specimens were rinsed with sterile deionized water and sterilized with 75% (v/v) ethanol for 0.5 h.They were then air-dried under ultraviolet (UV) light for 0.5 hours before use.
Marine Pseudomonas aeruginosa MCCC 1A00099 strain was purchased from Xiamen Marine Culture Collection Center (MCCC), China.Pseudomonas aeruginosa was grown aerobically at 37°C in 250 ml flasks and 500 ml electrochemical glass cells using Marine 2216E liquid medium (Qingdao Hope Biotechnology Co., Ltd., Qingdao, China).Medium (g/L): 19.45 NaCl, 5.98 MgCl2, 3.24 Na2SO4, 1.8 CaCl2, 0.55 KCl, 0.16 Na2CO3, 0.08 KBr, 0.034 SrCl2, 0.08 SrBr2, 0.022 H3BO3, 0.004 NaSiO3, 0016 NH3, 0016 NH3, 0016 NaH2PO4 , 5.0 peptone, 1.0 yeast extract and 0.1 ferric citrate.Autoclave at 121°C for 20 minutes before inoculation.Count sessile and planktonic cells using a hemocytometer under a light microscope at 400X magnification.The initial cell concentration of planktonic Pseudomonas aeruginosa immediately after inoculation was approximately 106 cells/ml.
Electrochemical tests were performed in a classic three-electrode glass cell with a medium volume of 500 ml.A platinum sheet and a saturated calomel electrode (SCE) were connected to the reactor via Luggin capillaries filled with salt bridges, serving as counter and reference electrodes, respectively.To make the working electrodes, a rubber-coated copper wire was attached to each specimen and covered with epoxy, leaving about 1 cm2 of exposed single-sided surface area for the working electrode.During electrochemical measurements, samples were placed in 2216E medium and maintained at a constant incubation temperature (37 °C) in a water bath.OCP, LPR, EIS and potential dynamic polarization data were measured using an Autolab potentiostat (Reference 600TM, Gamry Instruments, Inc., USA).LPR tests were recorded at a scan rate of 0.125 mV s-1 over the range of -5 and 5 mV with Eocp and a sampling frequency of 1 Hz.EIS was performed with a sine wave in the frequency range 0.01 to 10,000 Hz using a 5 mV applied voltage at steady state Eocp.Before the potential sweep, the electrodes were in open-circuit mode until a stable free corrosion potential value was reached.Polarization curves were then run from -0.2 to 1.5 V vs. Eocp at a scan rate of 0.166 mV/s.Each test was repeated 3 times with and without P. aeruginosa.
Specimens for metallographic analysis were mechanically polished with 2000 grit wet SiC paper and then further polished with 0.05 μm Al2O3 powder suspension for optical observation.Metallographic analysis was performed using an optical microscope.The specimens were etched with 10 wt.% potassium hydroxide solution 43.
After incubation, samples were washed 3 times with phosphate-buffered saline (PBS) solution (pH 7.4 ± 0.2) and then fixed with 2.5% (v/v) glutaraldehyde for 10 hours to fix biofilms.It was subsequently dehydrated with a graded series (50%, 60%, 70%, 80%, 90%, 95% and 100% v/v) of ethanol before air drying.Finally, the surface of the sample is sputtered with a gold film to provide conductivity for SEM observation.The SEM images were focused on the spots with the most sessile P. aeruginosa cells on the surface of each specimen.Perform EDS analysis to find chemical elements.A Zeiss Confocal Laser Scanning Microscope (CLSM) (LSM 710, Zeiss, Germany) was used to measure the pit depth.In order to observe the corrosion pits under the biofilm, the test piece was first cleaned according to the Chinese National Standard (CNS) GB/T4334.4-2000 to remove the corrosion products and biofilm on the surface of the test piece.
X-ray photoelectron spectroscopy (XPS, ESCALAB250 surface analysis system, Thermo VG, USA) analysis was performed using a monochromatic X-ray source (aluminum Kα line at 1500 eV energy and 150 W power) over a wide binding energy range 0 under standard conditions –1350 eV.High-resolution spectra were recorded using 50 eV pass energy and 0.2 eV step size.
The incubated specimens were removed and rinsed gently with PBS (pH 7.4 ± 0.2) for 15 s45.To observe the bacterial viability of the biofilms on the samples, the biofilms were stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, Eugene, OR, USA).The kit has two fluorescent dyes, a green fluorescent SYTO-9 dye and a red fluorescent propidium iodide (PI) dye.Under CLSM, dots with fluorescent green and red represent live and dead cells, respectively.For staining, a 1 ml mixture containing 3 μl SYTO-9 and 3 μl PI solution was incubated for 20 minutes at room temperature (23 oC) in the dark.Afterwards, the stained samples were observed at two wavelengths (488 nm for live cells and 559 nm for dead cells) using a Nikon CLSM machine (C2 Plus, Nikon, Japan).Biofilm thickness was measured in 3-D scanning mode.
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