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Microbial corrosion (MIC) is a serious problem in many industries, as it can lead to huge economic losses. Super duplex stainless steel 2707 (2707 HDSS) is used in marine environments due to its excellent chemical resistance. However, its resistance to MIC has not been experimentally demonstrated. This study examined the behavior of MIC 2707 HDSS caused by the marine aerobic bacterium Pseudomonas aeruginosa. Electrochemical analysis showed that in the presence of Pseudomonas aeruginosa biofilm in the 2216E medium, a positive change in the corrosion potential and an increase in the corrosion current density occur. Analysis of X-ray photoelectron spectroscopy (XPS) showed a decrease in the Cr content on the surface of the sample under the biofilm. Visual 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 completely immune to the MIC of P. aeruginosa biofilms.
Duplex stainless steels (DSS) are widely used in various industries due to the perfect combination of excellent mechanical properties and corrosion resistance1,2. However, localized pitting still occurs and affects the integrity of this steel3,4. DSS is not resistant to microbial corrosion (MIC)5,6. Despite the wide range of applications for DSS, there are still environments where the corrosion resistance of DSS is not sufficient for long-term use. This means that 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, in some cases, super duplex stainless steels (HDSS) with higher corrosion resistance are required. This led to the development of highly alloyed HDSS.
Corrosion resistance DSS depends on the ratio of alpha and gamma phases and depleted in Cr, Mo and W regions 8, 9, 10 adjacent to the second phase. HDSS contains a high content of Cr, Mo and N11, therefore it has excellent corrosion resistance and a high value (45-50) of the equivalent pitting resistance number (PREN) determined by wt.% Cr + 3.3 (wt.% Mo + 0.5 wt. .%W) + 16% wt. N12. Its excellent corrosion resistance depends on a balanced composition containing approximately 50% ferritic (α) and 50% austenitic (γ) phases. HDSS has better mechanical properties and higher resistance to chloride corrosion. Improved corrosion resistance extends the use of HDSS in more aggressive chloride environments such as marine environments.
MICs are a major problem in many industries such as the oil and gas and water industries14. MIC accounts for 20% of all corrosion damage15. MIC is a bioelectrochemical corrosion that can be observed in many environments. Biofilms that form on metal surfaces change the electrochemical conditions, thereby affecting the corrosion process. It is widely believed that MIC corrosion is caused by biofilms. Electrogenic microorganisms eat away metals to obtain the energy they need 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 intermediaries accelerate the transfer of electrons between Desulfovibrio sessificans cells and 304 stainless steel, resulting in more severe MIC attack. Anning et al. 19 and Wenzlaff et al. 20 have shown that biofilms of corrosive sulfate-reducing bacteria (SRBs) can directly absorb electrons from metal substrates, resulting in severe pitting.
DSS is known to be susceptible to MIC in media containing SRBs, iron-reducing bacteria (IRBs), etc. 21 . These bacteria cause localized pitting on the surface of DSS under biofilms22,23. Unlike DSS, the HDSS24 MIC is not well 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 elevated MIC concentrations. Pseudomonas is actively involved in the corrosion process and is recognized as a pioneer colonizer during biofilm formation. Mahat et al. 28 and Yuan et al. 29 demonstrated that Pseudomonas aeruginosa tends to increase the corrosion rate of mild steel and alloys in aquatic environments.
The main objective of this work was to investigate the properties of MIC 2707 HDSS caused by the marine aerobic bacterium Pseudomonas aeruginosa using electrochemical methods, surface analysis methods 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 behavior of the MIC 2707 HDSS. Energy dispersive spectrometric analysis (EDS) was carried out to detect chemical elements on a corroded surface. In addition, X-ray photoelectron spectroscopy (XPS) was used to determine the stability of oxide film passivation under the influence of a marine environment containing Pseudomonas aeruginosa. The depth of the pits was measured under a confocal laser scanning microscope (CLSM).
Table 1 shows the chemical composition of 2707 HDSS. Table 2 shows that 2707 HDSS has excellent mechanical properties with a yield strength of 650 MPa. On fig. 1 shows the optical microstructure of solution heat treated 2707 HDSS. In the microstructure containing about 50% austenite and 50% ferrite phases, elongated bands of austenite and ferrite phases without secondary phases are visible.
On fig. 2a shows the open circuit potential (Eocp) versus exposure time for 2707 HDSS in 2216E abiotic medium and P. aeruginosa broth for 14 days at 37°C. It shows that the largest and most significant change in Eocp occurs within the first 24 hours. The Eocp values ​​in both cases peaked at -145 mV (compared to SCE) around 16 h and then dropped sharply, reaching -477 mV (compared to SCE) and -236 mV (compared to SCE) for the abiotic sample. and P Pseudomonas aeruginosa coupons, respectively). After 24 hours, the Eocp 2707 HDSS value for P. aeruginosa was relatively stable at -228 mV (compared to SCE), while the corresponding value for non-biological samples was approximately -442 mV (compared to SCE). Eocp in the presence of P. aeruginosa was quite low.
Electrochemical study of 2707 HDSS samples 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 shows the electrochemical corrosion parameters of 2707 HDSS samples exposed to abiotic and Pseudomonas aeruginosa inoculated media over a period of 14 days. The tangents of the anode and cathode curves were extrapolated to obtain intersections giving corrosion current density (icorr), corrosion potential (Ecorr) and Tafel slope (βα and βc) according to standard methods30,31.
As shown in fig. 2b, an upward shift in the P. aeruginosa curve resulted in an increase in Ecorr compared to 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, which is four times greater than in the non-biological sample (0.087 µA cm-2).
LPR is a classic non-destructive electrochemical method for rapid corrosion analysis. It has also been used to study MIC32. On fig. 2c shows the polarization resistance (Rp) as a function of the exposure time. A higher Rp value means less corrosion. Within the first 24 hours, Rp 2707 HDSS peaked at 1955 kΩ cm2 for abiotic specimens and 1429 kΩ cm2 for Pseudomonas aeruginosa specimens. Figure 2c also shows that the Rp value decreased rapidly after one day and then remained relatively unchanged over the next 13 days. The Rp value of a Pseudomonas aeruginosa sample is about 40 kΩ cm2, which is much lower than the 450 kΩ cm2 value of a non-biological sample.
The value of icorr is proportional to the uniform corrosion rate. Its value can be calculated from the following Stern-Giri equation:
According to Zoe et al. 33, the typical value of the Tafel slope B in this work was taken to be 26 mV/dec. Figure 2d shows that the icorr of the non-biological sample 2707 remained relatively stable, while the P. aeruginosa sample fluctuated greatly after the first 24 hours. The icorr values ​​of P. aeruginosa samples were an order of magnitude higher than those of non-biological controls. This trend is consistent with the results of polarization resistance.
EIS is another non-destructive method used to characterize electrochemical reactions on corroded surfaces. Impedance spectra and calculated capacitance values ​​of samples exposed to abiotic environment and Pseudomonas aeruginosa solution, passive film/biofilm resistance Rb formed on the sample surface, charge transfer resistance Rct, electrical double layer capacitance Cdl (EDL) and constant QCPE Phase element parameters (CPE ). These parameters were further analyzed by fitting the data using an equivalent circuit (EEC) model.
On fig. 3 shows typical Nyquist plots (a and b) and Bode plots (a’ and b’) for 2707 HDSS samples in abiotic media 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 the increase in total impedance. Information about the relaxation time constant can be obtained from phase maxima. On fig. 4 shows the physical structures based on a monolayer (a) and a bilayer (b) and the 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 sample 2707 HDSS:
where Y0 is the KPI value, j is the imaginary number or (-1)1/2, ω is the angular frequency, n is the KPI power index less than one35. The charge transfer resistance inversion (ie 1/Rct) corresponds to the corrosion rate. The smaller Rct, the higher the corrosion rate27. After 14 days of incubation, the Rct of Pseudomonas aeruginosa samples reached 32 kΩ cm2, which is much less than the 489 kΩ cm2 of non-biological samples (Table 4).
The CLSM images and SEM images in Figure 5 clearly show that the biofilm coating on the surface of HDSS sample 2707 after 7 days is dense. However, after 14 days, biofilm coverage was poor and some dead cells appeared. Table 5 shows the biofilm thickness on 2707 HDSS samples 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 at 7 days, (b) 3-D CLSM image at 14 days, (c) SEM image at 7 days, and (d) SEM image at 14 days.
EMF revealed chemical elements in biofilms and corrosion products on samples exposed to P. aeruginosa for 14 days. On fig. Figure 6 shows that the content of C, N, O, and P in biofilms and corrosion products is significantly higher than in pure metals, since these elements are associated with biofilms and their metabolites. Microbes need only trace amounts of chromium and iron. High levels of Cr and Fe in the biofilm and corrosion products on the surface of the samples indicate that the metal matrix has lost elements due to corrosion.
After 14 days, pits with and without P. aeruginosa were observed in medium 2216E. Before incubation, the surface of the samples was smooth and defect-free (Fig. 7a). After incubation and removal of biofilm and corrosion products, the deepest pits on the surface of the samples were examined using CLSM, as shown in Fig. 7b and c. No obvious pitting was found on the surface of non-biological controls (maximum pitting depth 0.02 µm). The maximum pit depth caused by P. aeruginosa was 0.52 µm at 7 days and 0.69 µm at 14 days, based on the average maximum pit depth from 3 samples (10 maximum pit depths were selected for each sample). Achievement of 0.42 ± 0.12 µm and 0.52 ± 0.15 µm, respectively (Table 5). These hole depth values ​​are small but important.
(a) before exposure, (b) 14 days in an abiotic environment, and (c) 14 days in Pseudomonas aeruginosa broth.
On fig. Table 8 shows the XPS spectra of various sample surfaces, and the chemical composition analyzed for each surface is 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 non-biological controls. (samples C and D). For a P. aeruginosa sample, the spectral curve at the level of the Cr 2p nucleus was fitted to four peak components with binding energies (BE) 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 samples, the spectrum of the main Cr 2p level contains two main peaks for Cr (573.80 eV for BE) and Cr2O3 (575.90 eV for BE) in Figs. 9c and d, respectively. The most striking difference between abiotic samples and P. aeruginosa samples was the presence of Cr6+ and a higher relative proportion of Cr(OH)3 (BE 586.8 eV) under the biofilm.
The broad XPS spectra of the surface of sample 2707 HDSS in two media are 7 and 14 days, respectively.
(a) 7 days exposure to P. aeruginosa, (b) 14 days exposure to P. aeruginosa, (c) 7 days in an abiotic environment, and (d) 14 days in an abiotic environment.
HDSS exhibits a high level of corrosion resistance in most environments. Kim et al.2 reported that HDSS UNS S32707 was identified as a highly alloyed DSS with a PREN greater than 45. The PREN value of sample 2707 HDSS in this work was 49. This is due to the high chromium content and the high content of molybdenum and nickel, which are useful in acidic environments. and environments with high chloride content. In addition, a well-balanced composition and defect-free microstructure are beneficial 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 P. aeruginosa biofilm MICs.
Electrochemical results showed that the corrosion rate of 2707 HDSS in P. aeruginosa broth increased significantly after 14 days compared to the non-biological environment. In Figure 2a, a decrease in Eocp was observed both in the abiotic medium and in P. aeruginosa broth during the first 24 hours. After that, the biofilm completely covers the surface of the sample, and Eocp becomes relatively stable36. However, the biological Eocp level was much higher than the non-biological Eocp level. There are reasons to believe that this difference is associated with the formation of P. aeruginosa biofilms. On fig. 2d in the presence of P. aeruginosa, the icorr 2707 HDSS value reached 0.627 μA cm-2, which is 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, the impedance values ​​in the P. aeruginosa broth increased due to the attachment of P. aeruginosa cells and the formation of biofilms. However, when the biofilm completely covers the sample surface, the impedance decreases. The protective layer is attacked primarily due to the formation of biofilms and biofilm metabolites. Consequently, the corrosion resistance decreased over time and the attachment of P. aeruginosa caused localized corrosion. The trends in abiotic environments 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. In addition, for abiotic accessions, the Rct 2707 HDSS value reached 489 kΩ cm2 on day 14, which is 15 times higher than the Rct value (32 kΩ cm2) in the presence of P. aeruginosa. Thus, 2707 HDSS has excellent corrosion resistance in a sterile environment, but is not resistant to MICs from P. aeruginosa biofilms.
These results can also be observed from the polarization curves in Figs. 2b. Anodic branching has been associated with Pseudomonas aeruginosa biofilm formation and metal oxidation reactions. In this case, the cathodic reaction is the reduction of oxygen. The presence of P. aeruginosa significantly increased the corrosion current density, about an order of magnitude higher than in the abiotic control. This indicates that the P. aeruginosa biofilm enhances localized corrosion of 2707 HDSS. Yuan et al.29 found that the corrosion current density of the Cu-Ni 70/30 alloy increased under the action 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 2707 HDSS in this work. There may also be less oxygen under aerobic biofilms. Therefore, the refusal to re-passivate the metal surface with oxygen may be a factor contributing to MIC in this work.
Dickinson et al. 38 suggested that the rate of chemical and electrochemical reactions can be directly affected by the metabolic activity of sessile bacteria on the sample surface and the nature of the corrosion products. As shown in Figure 5 and Table 5, the number of cells and biofilm thickness decreased after 14 days. This can reasonably be explained by the fact 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, a P. aeruginosa biofilm contributed to local depletion of Cr and Fe under the biofilm on the surface of 2707 HDSS (Fig. 6). Table 6 shows the reduction in Fe and Cr in sample D compared to sample C, indicating that the dissolved Fe and Cr caused by the P. aeruginosa biofilm persisted for the first 7 days. The 2216E environment is used to simulate the marine environment. It contains 17700 ppm Cl-, which is comparable to its content in natural sea water. The presence of 17700 ppm Cl- was the main reason for the decrease in Cr in 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 resistance of 2707 HDSS to chlorine under abiotic conditions. On fig. 9 shows the presence of Cr6+ in the passivating film. It may be involved in the removal of chromium 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. Thus, below the P. aeruginosa biofilm, organic acid corrosion is unlikely to contribute 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 initial 7.4 to final 7.5) during the 14 day test period. The increase in pH in the inoculation medium after incubation was associated with 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 is much greater than that of the abiotic medium (0.02 µm). This is consistent with the electrochemical data described above. The pit depth of 0.69 µm is more than ten times smaller than the 9.5 µm value reported for 2205 DSS under the same conditions. These data show that 2707 HDSS exhibits better resistance to MICs than 2205 DSS. This should not come as a surprise since 2707 HDSS has higher Cr levels which provide longer passivation, more difficult to depassivate P. aeruginosa, and because of its balanced phase structure without harmful secondary precipitation causes pitting.
In conclusion, MIC pits were found on the surface of 2707 HDSS in P. aeruginosa broth compared to insignificant pits in the abiotic environment. This work shows that 2707 HDSS has better resistance to MIC than 2205 DSS, but it is not completely immune to MIC due to P. aeruginosa biofilm. These results assist in the selection of suitable stainless steels and life expectancy for the marine environment.
Coupon for 2707 HDSS provided by Northeastern University (NEU) School of Metallurgy 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 treated for solid solution at 1180°C for 1 hour. Prior to corrosion testing, a coin-shaped 2707 HDSS with a top open surface area of ​​1 cm2 was polished to 2000 grit with silicon carbide sandpaper and then polished with a 0.05 µm Al2O3 powder slurry. The sides and bottom are protected with inert paint. After drying, the samples were washed 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 h before use.
Marine Pseudomonas aeruginosa strain MCCC 1A00099 was purchased from the Xiamen Marine Culture Collection Center (MCCC), China. Pseudomonas aeruginosa was grown under aerobic conditions at 37° C. in 250 ml flasks and 500 ml glass electrochemical cells using Marine 2216E liquid medium (Qingdao Hope Biotechnology Co., Ltd., Qingdao, China). Medium contains (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 6NH26NH3, 3.0016 NH3 5.0 peptone, 1.0 yeast extract and 0.1 iron citrate. Autoclave at 121°C for 20 minutes prior to inoculation. Count sessile and planktonic cells with a hemocytometer under a light microscope at 400x magnification. The initial concentration of planktonic Pseudomonas aeruginosa immediately after inoculation was approximately 106 cells/ml.
Electrochemical tests were carried out in a classic three-electrode glass cell with a medium volume of 500 ml. The platinum sheet and saturated calomel electrode (SAE) were connected to the reactor through Luggin capillaries filled with salt bridges, which served as counter and reference electrodes, respectively. For the manufacture of working electrodes, rubberized copper wire was attached to each sample and covered with epoxy resin, leaving about 1 cm2 of unprotected area for the working electrode on one side. During electrochemical measurements, the samples were placed in the 2216E medium and kept 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 in the range of -5 to 5 mV with Eocp and a sampling rate of 1 Hz. EIS was performed with a sine wave over a frequency range of 0.01 to 10,000 Hz using an applied voltage of 5 mV at steady state Eocp. Before the potential sweep, the electrodes were in idle mode until a stable value of the free corrosion potential was reached. The polarization curves were then measured from -0.2 to 1.5 V as a function of Eocp at a scan rate of 0.166 mV/s. Each test was repeated 3 times with and without P. aeruginosa.
Samples for metallographic analysis were mechanically polished with wet 2000 grit SiC paper and then further polished with a 0.05 µm Al2O3 powder suspension for optical observation. Metallographic analysis was performed using an optical microscope. The samples were etched with a 10 wt% solution of potassium hydroxide 43.
After incubation, the samples were washed 3 times with phosphate buffered saline (PBS) (pH 7.4 ± 0.2) and then fixed with 2.5% (v/v) glutaraldehyde for 10 hours to fix biofilms. It was then dehydrated with batched ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100% by volume) before air drying. Finally, a gold film is deposited onto the surface of the sample to provide conductivity for SEM observation. SEM images were focused on spots with the most sessile P. aeruginosa cells on the surface of each sample. Perform an EDS analysis to find chemical elements. A Zeiss confocal laser scanning microscope (CLSM) (LSM 710, Zeiss, Germany) was used to measure the pit depth. To observe corrosion pits under the biofilm, the test sample was first cleaned according to the Chinese National Standard (CNS) GB/T4334.4-2000 to remove corrosion products and biofilm from the surface of the test sample.
X-ray photoelectron spectroscopy (XPS, ESCALAB250 surface analysis system, Thermo VG, USA) analysis was performed using a monochromatic X-ray source (Aluminum Kα line with an energy of 1500 eV and a power of 150 W) in a wide range of binding energies 0 under standard conditions of –1350 eV. High resolution spectra were recorded using a transmission energy of 50 eV and a step of 0.2 eV.
The incubated samples were removed and washed gently with PBS (pH 7.4 ± 0.2) for 15 s45. To observe bacterial viability of biofilms on samples, biofilms were stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, Eugene, OR, USA). The kit contains two fluorescent dyes: SYTO-9 green fluorescent dye and propidium iodide (PI) red fluorescent dye. In CLSM, fluorescent green and red dots represent live and dead cells, respectively. For staining, 1 ml of a mixture containing 3 µl of SYTO-9 and 3 µl of PI solution was incubated for 20 minutes at room temperature (23°C) in the dark. Thereafter, the stained samples were examined at two wavelengths (488 nm for live cells and 559 nm for dead cells) using a Nikon CLSM apparatus (C2 Plus, Nikon, Japan). The biofilm thickness was measured in 3D scanning mode.
How to cite this article: Li, H. et al. Microbial corrosion of 2707 super duplex stainless steel by Pseudomonas aeruginosa marine biofilm. the science. 6, 20190. doi: 10.1038/srep20190 (2016).
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