Microbiologically Influenced Corrosion of 2707 Hyper-Duplex Stainless Steel by Marine Pseudomonas aeruginosa Biofilm.

Microbiologically Influenced Corrosion (MIC) is a serious problem in many industries because it causes huge economic losses. Due to its excellent resistance to chemical corrosion, 2707 hyper duplex stainless steel (2707 HDSS) has been used in the marine environment. However, its resistance to MIC was not experimentally proven. In this study, the MIC behavior of 2707 HDSS caused by the marine aerobe Pseudomonas aeruginosa was investigated. Electrochemical analyses demonstrated a positive shift in the corrosion potential and an increase in the corrosion current density in the presence of the P. aeruginosa biofilm in the 2216E medium. X-ray photoelectron spectroscopy (XPS) analysis results showed a decrease in Cr content on the coupon surface beneath the biofilm. The pit imaging analysis showed that the P. aeruginosa biofilm caused a largest pit depth of 0.69 μm in 14 days of incubation. Although this was quite small, it indicated that 2707 HDSS was not completely immune to MIC by the P. aeruginosa biofilm.

Duplex n class="Chemical">stainless steelpan> (n class="Chemical">DSS) is widely used in various industries due to its desirable combination of excellent mechanical properties and corrosion resistance12. However, localized pitting corrosion can still ocn class="Chemical">cur and it affects the integrity of this kind of steel34. DSS is not immune to microbiologically influenced corrosion (MIC)56. Despite a very broad range of applications for DSS, there are still environments where the corrosion resistance of DSS is inadequate for long-term services. This means, more expensive materials with higher corrosion resistance are needed. Jeon et al.7 found that even super duplex stainless steels (SDSSs) exhibit some limits in corrosion resistance. Therefore, hyper duplex stainless steels (HDSSs) with a higher corrosion resistance are needed in some applications. This led to the development of highly alloyed HDSSs.

The corrosion resistapan class="Chemical">nce of n class="Chemical">DSS is determinpan>ed by the ratio of the α-phase anclass="Chemical">pan>d γ-phase and by the pan> class="Chemical">Cr, Mo, and W depleted regions that are adjacent to the secondary phases8910. HDSS contains high levels of n class="Chemical">Cr, Mo and N11, resulting in its excellent corrosion resistance and a high value (45–50) of pitting resistance equivalent number (PREN), which is calculated from wt.% Cr + 3.3 (wt.% Mo + 0.5 wt.% W) + 16 wt.% N12. Its excellent corrosion resistance properties rely on a well-balanced composition with approximately 50% ferrite (α) and 50% austenite (γ) phases, that offer HDSS improved mechanical properties and higher chloride corrosion resistance compared with conventional DSS13. The improved corrosion resistance extends the use of HDSS in more aggressive chloride environments, such as marine environments.

MIC is a major problem in many industries such as n class="Chemical">oil anpan>d gas, as well as pan> class="Chemical">water utilities14. MIC accounts for 20% of all corrosion damages15. MIC is bioelectrochemical corrosion that can be observed in many environments16. Biofilms formed on a metal surface will change the electrochemical conditions and thus influencing the corrosion processes. It is widely accepted that MIC corrosion is caused by biofilms14. Electrogenic microbes corrode metals in order to obtain maintenance energy for survival17. The most recent MIC research suggested that EET (extracellular electron transfer) is a rate-limiting factor in MIC caused by electrogenic microbes. Zhang et al.18 demonstrated that an electron mediator accelerated the electron transfer between sessile Desulfovibrio vulgaris cells and 304 stainless steel, resulting in a much more severe MIC attack. Enning et al.19 and Venzlaff et al.20 showed that a corrosive sulfate-reducing bacterium (SRB) biofilm was capable of directly uptaking electrons from the metal matrix, resulting in serious pitting corrosion.

n class="Chemical">DSS is known to be susceptible to MIC in enpan>vpan> class="Chemical">ironments containing SRB, iron reducing bacteria (IRB), etc21. These bacteria cause localized pitting corrosion on DSS surfaces under biofilms2223. Unlike DSS, very little is known for the MIC of Hn class="Chemical">DSS24.

n class="Species">Pseudomonas aeruginosa is a Gram-negative motile rod bacterium widely distributed in nature25. pan> class="Species">P. aeruginosa is also a predominant group of micro-organisms in marine environments causing MIC to steels26. The genus Pseudomonas is closely involved in corrosion processes and has been recognized as the pioneer colonizer in the process of biofilm formation27. Mahat et al.28 and Yuan et al.29 demonstrated that P. aeruginosa has the propensity to increase the corrosion rates of mild steels and alloys in aquatic environments.

The main aim of this work was to investigate the MIC characteristics of 2707 Hn class="Chemical">DSS caused by the marinpan>e aerobe class="Chemical">pan> class="Species">P. aeruginosa using electrochemical methods, surface analysis techniques and corrosion product analyses. 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. The energy dispersive spectrometer (EDS) analysis was undertaken to find the chemical elements on the corroded surface. In addition, the X-ray photoelectron spectroscopy (XPS) analysis was used to determine the stability of the oxide film passivity under the influence of a marine environment containing P. aeruginosa. The pit depth was measured under a confocal laser scanning microscope (CLSM).

Results

Material characterizations

Table 1 lists the chemical composition of 2707 Hn class="Chemical">DSS. Table 2 shows that 2707 Hclass="Chemical">pan> class="Chemical">DSS has excellent mechanical properties with yield strength of 650 MPa. Figure 1 reveals the optical microstructures of the solution heat-treated 2707 HDSS. Elongated bands of austenite and ferrite phases without secondary phases can be seen in the microstructure that contains approximately 50% austenite and 50% ferrite phases.

Table 1

Chemical composition of 2707 HDSS.

Material	Element
	Si	Mn	P	S	Co	Cr	Ni	Mo	Cu	N	Fe
2707 HDSS (wt.%)	0.42	1.11	0.005	0.003	1.00	26.83	7.14	4.88	0.98	0.39	Bal.

Table 2

Mechanical properties of 2707 HDSS.

Material	Tensile strength (MPa)	Yield strength 0.2% (MPa)	Elongation (%)
2707 HDSS	950	650	25

Figure 1

Microstructure of 2707 HDSS:

(A) ferrite, and (B) austenite.

Open circuit potential versus time

Figure 2a shows open cirn class="Chemical">cuit potenclass="Chemical">pan>tial (n class="Chemical">Eocp) vs. exposure time data for 2707 Hn class="Chemical">DSS in the abiotic 2216E medium and in the P. aeruginosa broth at 37 °C for 14 days. It reveals that the most and significant variation in Eocp occurred in the initial 24 h. The Eocp values in both cases peaked around −145 mV (vs. SCE) at about 16 h and then dropped sharply, reaching −477 mV (vs. SCE) and −236 mV (vs. SCE) for the abiotic coupon and the P. aeruginosa coupon, respectively. After 24 h, the Eocp value of 2707 HDSS remained relatively stable at −228 mV (vs. SCE) for the P. aeruginosa coupon, while the corresponding value for the abiotic coupon was approximately −442 mV (vs. SCE). The Eocp in the presence of P. aeruginosa was considerably lower.

Figure 2

Electrochemical tests for 2707 HDSS coupons in the abiotic medium and in the P. aeruginosa broth at 37 °C:

(a) variation of Eocp with exposure time, (b) polarization curves on the 14th day, (c) variation of Rp with exposure time, and (d) variation of icorr with exposure time.

Polarization Curves

The values of the electrochemical corrosion parameters of 2707 Hpapan class="Chemical">n class="Chemical">DSS couponclass="Chemical">pan>s after exposure for 14 days in the abiotic medium as well as inpan> the pan> class="Species">P. aeruginosa inoculated medium are listed in Table 3. The tangential lines of the anodic and cathodic curves were extrapolated to reach an intersection point to yield corrosion n class="Chemical">current density (icorr), corrosion potential (Ecorr), and Tafel slopes (βα and βc) following the standard approach3031.

Table 3

The electrochemical corrosion parameters determined from the polarization curves of 2707 HDSS after 14 days of incubation at 37 oC.

Medium	icorr (μA cm−2)	Ecorr (mV)	βa (mV/dec)	βc (mV/dec)
Sterile	0.087	−472	286.4	−237.8
P. aeruginosa inoculated	0.328	−277	121.2	−153.6

As shown in Figure 2b, compared with the abiotic curves, the P. aeruginosa curves shifted upward leading to an increase of Ecorr. The icorr value, that was directly proportional to the corrosion rate, increased to 0.328 μA cm−2 for the P. aeruginosa coupon, four times greater than that for the abiotic coupon (0.087 μA cm−2).

Linear polarization resistance

LPR is a classical electrochemical method for fast corrosion analysis that is non-destructive. It is also used to study MIC32. Figure 2c shows the variation of the polarization resistance (Rp) as a function of exposure time. A higher Rp value means less corrosion. During the initial 24 h, the Rp of 2707 Hn class="Chemical">DSS reached a maximum value of 1955 kΩ cm2 for the abiotic couponpan> anpan>d 1429 kΩ cm2 for the pan> class="Species">P. aeruginosa coupon. Figure 2c also shows that the Rp value fell quickly after one day and then remained relatively unchanged for the next 13 days. The Rp value for the P. aeruginosa coupon was around 40 kΩ cm2, much lower than the 450 kΩ cm2 value for the abiotic coupon.

The icorr value is directly proportional to the upan class="Chemical">niform corrosion rate. Its value can be caln class="Chemical">culated from the Stern-Geary equation below,

Following Zou et al.33, the Tan class="Chemical">fel slope B in this work was assumed a typical value of 26 mV/dec. Figure 2d shows that icorr remained relatively stable for the abiotic 2707 couponpan> while it fluctuated conpan>siderably for the pan> class="Species">P. aeruginosa coupon that had a large jump after the initial 24 h. The icorr value for the P. aeruginosa coupon was one order of magnitude higher than that of the abiotic control. This trend agreed with the polarization resistance result.

Electrochemical impedance spectroscopy

EIS is another nondestructive technique to characterize electrochemical reactions at a corrosion interface34. Impedance spectra and caln class="Chemical">culated capacitanpan>ce values of couponpan>s exposed to the abiotic medium anpan>d the pan> class="Species">P. aeruginosa solution, Rb the resistance of passive film/biofilm formed on the coupon surface, Rct the charge transfer resistance, Cdl the capacitance of the electrical double layer (EDL), and QCPE the constant phase element (CPE) parameter. These parameters were further analyzed by fitting data with an equivalent electrical circuit (EEC) model.

Figure 3 shows the typical n class="Chemical">Nyquist diagrams (a and b) anpan>d Bode plots (a′ anpan>d b′) at difn class="Chemical">ferent incubation times for 2707 HDSS coupons in the abiotic medium and in the P. aeruginosa broth. The diameters of the n class="Chemical">Nyquist loops decreased in the presence of P. aeruginosa. The Bode plot (Figure 3b′) shows an increase of the total impedance magnitudes. The information of the relaxation time constants can be provided by the phase maxima. Figure 4 shows the physical structure and their corresponding EECs based on a single-layer (a) and a double-layer (b). CPE was introduced into the EEC models. Its admittance and impedance, respectively, are expressed below:

Figure 3

The Nyquist and Bode plots of 2707 HDSS coupons with and without exposure to P. aeruginosa:

(a,a′) 2707 HDSS in the abiotic medium, and (b,b′) 2707 HDSS in the P. aeruginosa broth.

Figure 4

Two physical models and the corresponding equivalent circuits used for fitting the impedance spectra of 2707 HDSS coupons:

(a) in the abiotic medium, and (b) in the P. aeruginosa broth.

where Y0 is the magnitude of the CPE, j the imaginary number or (−1)1/2, ω the angular frequency, and n the CPE power index which is less than unity35. The inverse of the charge-transn class="Chemical">fer resistanpan>ce (i.e., 1/Rct) corresponpan>ds to the corrosionpan> rate. A smaller Rct meanpan>s a faster corrosionpan> rate27. After 14 days of inpan> class="Chemical">cubation, the Rct of the P. aeruginosa coupon reached 32 kΩ cm2, much smaller than the 489 kΩ cm2 for the abiotic coupon (Table 4).

Table 4

Electrochemical model impedance parameters of 2707 HDSS in the P. aeruginosa broth and in the abiotic medium.

Duration(days)	Rs(Ω cm2)	QCPE×10−5(Ω−1 Sn cm−2)	n	Rb(kΩ cm2)	Cdl(μF cm−2)	Rct(kΩ cm2)
2707 HDSS in the P. aeruginosa broth
1	9.46	6.416	0.7961	16.48	168.4	24.9
4	8.56	7.791	0.7702	21.65	274.6	29.2
7	9.49	6.476	0.7539	21.68	185.9	29.1
14	7.93	6.446	0.7016	24.61	103.1	32.1
2707 HDSS in the abiotic medium
1	11.89	4.360	0.8676	—	—	807.2
4	11.01	3.224	0.8405	—	—	388.0
7	11.75	2.979	0.8273	—	—	568.3
14	9.56	2.720	0.7803	—	—	489.4

Biofilm visualization

The CLSM images and SEM images ipan class="Chemical">n Figure 5 clearly indicate that the biofilm coverage on the 2707 Hn class="Chemical">DSS couponpan> surface after 7 days was denpan>se. However, after 14 days the biofilm coverage was sparse anpan>d some dead cells appeared. Table 5 shows the biofilm thicknclass="Chemical">pan>ess on the 2707 Hpan> class="Chemical">DSS coupon after exposure to P. aeruginosa for 7 and 14 days. The largest biofilm thickness changed from 23.4 μm after 7 days to 18.9 μm after 14 days. The average biofilm thickness also confirmed the trend. It den class="Chemical">creased from 22.2 ± 0.7 μm after 7 days to 17.8 ± 1.0 μm after 14 days.

Figure 5

The morphology of P. aeruginosa biofilms on coupon surfaces:

(a) the 3-D CLSM image after 7 days, (b) the 3-D CLSM image after 14 days, (c) the SEM image after 7 days, and (d) the SEM image after 14 days.

Table 5

Pit depth and biofilm thickness of 2707 HDSS in the abiotic medium and P. aeruginosa broth.

	abioticmedium	P. aeruginosaincubated for 7 days	P. aeruginosaincubated for 14 days
Maximum pit depth (μm)	0.02	0.52	0.69
Average largest pit depth (μm)	0.017 ± 0.01	0.42 ± 0.12	0.52 ± 0.15
Largest biofilm thickness (μm)	—	23.4	18.9
Average biofilm thickness (μm)	—	22.2 ± 0.7	17.8 ± 1.0

Chemical elements in the biofilm and corrosion products on the coupon exposed to n class="Species">P. aeruginosa for 14 days were revealed by EDS. Figure 6 shows the amounts of C, pan> class="Chemical">N, O, P in the biofilm and corrosion products were much higher than those in the bare metal because these elements were associated with the biofilm and its metabolic products. Cr and Fe are needed by microorganisms only at trace levels. The high levels of Cr and Fe in the biofilm and corrosion products on the coupon surface suggest loss of the elements by the metal matrix due to corrosion.

Figure 6

Results of EDS analysis of the 2707 HDSS surface after exposure to P. aeruginosa for 14 days.

Pitting corrosion was observed with and without n class="Species">P. aeruginosa inpan> the 2216E medium after 14 days. Before inpan>pan> class="Chemical">cubation, the coupon surfaces were smooth and had no defects (Figure 7a). After incubation and removing the biofilm and corrosion products, the deepest pits on the coupon surfaces were examined under CLSM as shown in Figure 7b and c. No significant pits were found on the abiotic control coupon surface (largest pit depth 0.02 μm). The largest pit depth caused by P. aeruginosa was 0.52 μm after 7 days and 0.69 μm after 14 days, and the average largest pit depth based on 3 coupons (with 10 largest pit depth values per coupon selected) reached 0.42 ± 0.12 μm and 0.52 ± 0.15 μm, respectively (Table 5). These pit depth values were small, but significant.

Figure 7

CLSM analysis of pits on coupon surfaces:

(a) before exposure, (b) in the abiotic medium for 14 days, and (c) in the P. aeruginosa broth for 14 days.

XPS analysis of 2707 HDSS coupon surfaces

Figure 8 shows the XPS spectra of difpapan class="Chemical">n class="Chemical">ferenclass="Chemical">pan>t coupon surfaces, anpan>d the chemical compositionpan> of each surface anpan>alysis is summarized in Table 6. Inpan> Table 6, the atomic percenpan>tages of pan> class="Chemical">Fe and Cr in the presence of P. aeruginosa (samples A and B) were much lower than those for the abiotic control coupons (samples C and D). For the P. aeruginosa coupons, the Cr 2p core-level spectrum was curve-fitted into four peak components at binding energy (BE) values of 574.4, 576.6, 578.3 and 586.8 eV, which were attributable to Cr, Cr2O3, n class="Chemical">CrO3 and Cr(OH)3, respectively (Figure 9a and b). For the abiotic coupons, the Cr 2p core-level spectrum contained two dominant peaks for Cr (BE at 573.80 eV) and Cr2O3 (BE at 575.90 eV) in Figure 9c and d, respectively. The most noticeable difference between the abiotic coupons and the P. aeruginosa coupons was the presence of Cr6+ beneath the biofilm and a higher relative portion of Cr(OH)3 (BE at 586.8 eV).

Figure 8

The wide XPS spectra for the surfaces of 2707 HDSS coupons in the both media for 7 days and 14 days, respectively.

Table 6

Relative atomic percent (%) of the main constituents on the surface of 2707 HDSS with and without exposure to P. aeruginosa: (A) in the abiotic medium for 7 days, (B) in the abiotic medium for 14 days, (C) in the inoculated medium for 7 days, and (D) in the inoculated medium for 14 days.

Sample	Atomic percent (%)
	Cl	C	N	O	Cr	Fe	Na	Mg
A	4.59	38.81	5.45	36.67	3.69	1.82	7.78	0.22
B	7.39	42.46	5.30	29.64	2.95	1.30	9.65	0.75
C	6.39	58.93	5.76	23.70	0.51	0.39	3.56	0.43
D	2.62	56.95	5.35	28.71	0.21	0.16	5.64	0.36

Figure 9

The high resolution XPS spectra of Cr 2p for 2707 HDSS:

(a) with exposure to P. aeruginosa for 7 days, (b) with exposure to P. aeruginosa for 14 days, (c) in the abiotic medium for 7 days, and (d) in the abiotic medium for 14 days.

Discussion

Hn class="Chemical">DSSpan>s exhibit a high level of corrosion resistanpan>ce inpan> most enpan>vpan> class="Chemical">ironments. Kim et al.2 reported that UNS S32707 HDSS is defined as a highly alloyed DSS with a PREn class="Chemical">N in excess of 45. The 2707 HDSS coupons in this work had a PREN value of 49. This is due to its high Cr content and elevated Mo and Ni levels, which are beneficial in acid and high chloride environments. Furthermore, the well-balanced composition and defect-free microstructure are useful for structural stability and corrosion resistance. Despite its superior chemical corrosion resistance, however, the experimental data in this work demonstrated that 2707 HDSS was not completely immune to the MIC by the P. aeruginosa biofilm.

The electrochemical results revealed that the corrosion rate of 2707 Hn class="Chemical">DSS signclass="Chemical">pan>ificantly inpan>pan> class="Chemical">creased in the P. aeruginosa broth after 14 days compared to the abiotic medium. In Figure 2a, a decrease of n class="Chemical">Eocp was observed in both the abiotic medium and the P. aeruginosa broth in the initial 24 h. Afterwards, the biofilm completed the coverage of the coupon surface, Eocp became relatively stable36. However, the biotic Eocp was at a much higher level than the abiotic Eocp. It is reasonable to believe that the difference was due to the formation of the P. aeruginosa biofilm. In Figure 2d, the icorr value of 2707 HDSS reached 0.627 μA cm−2 in the presence of P. aeruginosa, one order of magnitude higher than that for the abiotic control (0.063 μA cm−2), which was consistent with the Rct values measured by EIS. In the first few days, the impedance value increased in the P. aeruginosa broth due to the attachment of P. aeruginosa cells and the formation of the biofilm. However, the impedance then decreased when the biofilm fully covered the coupon surface. The protective layer was first attacked because of the formation of the biofilm and metabolites of the biofilm. Consequently, the corrosion resistance decreased with time and the P. aeruginosa attachment caused the localized corrosion. The tendency in the abiotic medium was different. The corrosion impedance of the abiotic control was much higher than the corresponding value of the coupon exposed to the P. aeruginosa broth. Furthermore, for the abiotic samples, the Rct value of 2707 HDSS reached 489 kΩ cm2 on the 14th day, fifteen times larger than that in the presence of P. aeruginosa (32 kΩ cm2). Thus, 2707 HDSS possessed excellent corrosion resistance in the sterile environment, but it was not immune to the MIC attack by the P. aeruginosa biofilm.

These results were also observed from the polarization papan class="Chemical">n class="Chemical">curves inpan> Figure 2b. The n class="Disease">anodic branch was attributed to the formation of n class="Species">P. aeruginosa biofilm and the metal oxidation reaction. Meanwhile the cathodic reaction was the reduction of oxygen. The presence of P. aeruginosa greatly increased the corrosion current density, which was approximately one order of magnitude higher than the abiotic control. It suggested that the P. aeruginosa biofilm increased the localized corrosion of 2707 HDSS. Yuan et al.29 found that the 70/30 Cu-Ni alloy corrosion current density increased under the attack of a P. aeruginosa biofilm. It could be due to the P. aeruginosa biofilm’s biocatalysis of the reduction of oxygen37. This observation could explain the MIC of 2707 HDSS in this work as well. It was also possible that the aerobic biofilm made oxygen less available underneath it. Thus, the failure to repassivate the metal surface by oxygen could be a contributing factor in the MIC in this work.

Dickinson et al.38 proposed that the rates of chemical and electrochemical reactions can be directly afn class="Chemical">fected by the metabolic activities of the sessile bacteria onpan> a couponpan> surface anpan>d the nature of corrosionclass="Chemical">pan> products. As seen inpan> Figure 5 anpan>d Table 5, both the cell number anpan>d biofilm thicknpan>ess depan> class="Chemical">creased after 14 days. This can be reasonably explained that after 14 days, most of the sessile cells on the 2707 HDSS surface were dead due to the exhaustion of the nutrients in the 2216E medium or the toxic metal ions released from the 2707 HDSS matrix. This was a limitation of batch experiments.

In this work, the papan class="Chemical">n class="Species">P. aeruginosa biofilm promoted the localized depletionpan> of class="Chemical">pan> class="Chemical">Cr and Fe underneath the biofilm on the 2707 HDSS surface (Figure 6). In Table 6, compared with sample C, there was a decrease of Fe and Cr in sample D, indicating that dissolution Fe and Cr caused by the P. aeruginosa biofilm continued beyond the first 7 days. The 2216E medium was used to simulate the marine environment. It contained 17700 ppm Cl−, comparable to that in the natural seawater. The presence of 17700 ppm Cl− was the main reason responsible for the decrease of Cr from the 7-day and 14-day abiotic samples analyzed by XPS. Compared with the P. aeruginosa coupons, the dissolution of Cr in abiotic coupons was much smaller due to the strong Cl− resistance of 2707 HDSS in an abiotic environment. Figure 9 shows that the presence of Cr6+ in the passivation film. It might be involved in the removal of Cr from the steel surface by the P. aeruginosa biofilm as suggested by Chen and Clayton39.

The pH values of the n class="Chemical">cupan>lture medium before and after inpan> class="Chemical">cubation were 7.4 and 8.2, respectively, due to bacterial growth. Thus, underneath the P. aeruginosa biofilm, organic acid corrosion was unlikely a contributing factor in this work due to the relatively high pH values in the bulk medium. The pH value of the abiotic control medium didn’t change significantly (from the initial 7.4 to the final 7.5) in the 14-day test duration. The inn class="Chemical">crease of pH in the inoculated medium after incubation was due to the metabolic activities of P. aeruginosa, which was found to have the same impact on pH in the absence of coupons.

As shown in Figure 7, the maximum pit depth caused by the n class="Species">P. aeruginosa biofilms was 0.69 μm, which was much larger thanpan> that in the abiotic medium (0.02 μm). This was conclass="Chemical">pan>sistent with the electrochemical data above. The 0.69 μm pit depth was more thanpan> tenpan> times smaller thanpan> the 9.5 μm value pan> class="Gene">reported for 2205 DSS under the same conditions40. These data proved that 2707 HDSS exhibited a better MIC resistance compared with 2205 DSS. This should not be a surprise because 2707 HDSS has a higher n class="Chemical">Cr level that offers more durable passivity, making it more difficult for P. aeruginosa to depassivate and initiate pitting corrosion due to the balanced phase structure without harmful secondary precipitates41.

In conclusion, MIC pitting corrosion was found on the surface of 2707 Hn class="Chemical">DSS inpan> the P. pan> class="Disease">aeruginosa broth compared to negligible pitting in the abiotic medium. This work demonstrated that 2707 HDSS had much better MIC resistance than 2205 DSS, but was not completely immune to MIC due to the P. aeruginosa biofilm. These findings are useful in the selection of a suitable stainless steel for the marine environment and in the estimation of service life span.

Methods

Coupon preparation

The coupons of 2707 Hpapan class="Chemical">n class="Chemical">DSS were provided by School of class="Chemical">pan> class="Chemical">Metallurgy, Northeastern University (NEU) in Shenyang, China. The elemental composition of 2707 HDSS was shown in Table 1, which was analyzed by the Department of Materials Analysis and Testing of NEU. All specimens were solution-treated at 1180 °C for 1 h. Prior to the corrosion tests, coin-shaped 2707 HDSS with a top exposed surface area of 1 cm2 were polished to 2000 grit with silicon carbide papers, and then further polished with a 0.05 μm Al2O3 powder suspension. The side and bottom surface were protected by an inert paint. After drying, the coupons 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 h before use.

Bacterial strain and cultivation

The marine papan class="Chemical">n class="Species">P. aeruginosa MCCC 1A00099 strain was purchased from the Marinpan>e n class="Chemical">Culture Collection of China (MCCC), Xiamen, China. The marine 2216E liquid medium (Qingdao Hope Bio-technology Co., Qingdao, China) was used to n class="Chemical">culture the P. aeruginosa at 37 °C aerobically in 250 ml flasks and in a 500 ml electrochemical glass cell. The culture medium contained (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, 0.0024 NaF, 0.0016 NH4NO3, 0.008 NaH2PO4, 5.0 peptone, 1.0 yeast extract, and 0.1 ferric citrate. It was autoclave-sterilized at 121 °C for 20 min prior to inoculation. A hemocytometer under a light microscope at 400× magnification was used to enumerate sessile and planktonic cells. The initial planktonic P. aeruginosa cell concentration immediately after inoculation was approximately 106 cells/ml.

Electrochemical test procedures

Electrochemical tests were carried out in a classical three-electrode glass cell with a n class="Chemical">culture medium volume of 500 ml. A class="Chemical">pan> class="Chemical">platinum sheet and a saturated calomel electrode (SCE) connected to the reactor via a Luggin capillary filled with a salt bridge as the counter and reference electrodes, respectively. To create a working electrode, a rubber-coated copper wire was connected to each coupon and covered with epoxy resin leaving an exposed one-sided surface area of approximately 1 cm2 for this working electrode. During electrochemical measurements, samples were placed in the 2216E medium, and maintained at a constant incubation temperature (37 °C) in a water bath. The OCP, LPR, EIS and potential dynamic polarization data were measured with 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 and 5 mV versus Eocp, and the sampling frequency was 1 Hz. EIS was performed under a steady-state Eocp using a 5 mV applied voltage at sinusoidal wave in a frequency range of 0.01 to 10,000 Hz. Before the potential scan, the electrodes were in the open circuit mode until a steady free corrosion potential value reached42. The polarization curves were then run at a scan rate of 0.166 mV/s from −0.2 to 1.5 V vs. Eocp. Each test was repeated three times with and without P. aeruginosa.

Surface analysis

The coupons for the papan class="Chemical">n class="Chemical">metallographic anclass="Chemical">pan>alysis were mechanically polished with wet SiC paper of 2000 grit, anpan>d thenpan> further polished with a 0.05 μm pan> class="Chemical">Al2O3 powder suspension to enable optical observations to be made. Metallographic analysis was carried out using an optical microscopy. The coupons were etched with a 10 wt.% potassium hydroxide solution43.

After inpapan class="Chemical">n class="Chemical">cubationclass="Chemical">pan>, a coupon was washed with a pan> class="Chemical">phosphate buffer n class="Chemical">saline (PBS) solution (pH 7.4 ± 0.2) three times and then fixed with 2.5% (v/v) glutaraldehyde for 10 h to fix the biofilm. 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 coupon surface was sputter-coated with a gold film to provide conductivity for SEM observation44. SEM images focused on spots with the most sessile P. aeruginosa cells on each coupon surface. The EDS analysis was undertaken to find the chemical elements. A Zeiss confocal laser scanning microscope (CLSM) (LSM 710, Zeiss, Germany) was used to measure the pit depth. To observe the corrosion pits underneath a biofilm, the coupon was first cleaned according to the Chinese National Standards (CNS) GB/T4334.4-2000 to remove the corrosion products and biofilms on the coupon surface.

An X-ray photoelectron spectroscopy (XPS, ESCALAB250 surface analysis system, Thermo VG, USA) analysis was performed using a monochromatic X-ray source (an aluminum Kα line of 1500 eV energy and 150 W power) within the wide binding energy range of 0–1350 eV under standard conditions. The high resolution spectra were recorded using 50 eV pass energy and 0.2 eV step.

Bacterial stain assay

The coupons after inn class="Chemical">cubationpan> were takenclass="Chemical">pan> out and genpan>tly rinsed with PBS (pH 7.4 ± 0.2) for 15 s45. Inpan> order to observe the bacterial viability of the biofilms onpan> the couponpan>, LIVE/DEAD BacLight Bacterial Viability Kits (Inpan>vitrogenpan>, Eugenpan>e, OR, USA) were used to stain the biofilms. The kits have two fluorescenpan>t dyes, the greenpan>-fluorescenpan>t pan> class="Chemical">SYTO-9 dye and the red-fluorescent propidium iodide (PI) dye. Under CLSM, the dots with fluorescent green and red represented the live and dead cells, respectively. When staining, 1 ml mixture containing 3 μl SYTO-9 and 3 μl PI solutions were incubated for 20 min in the dark at room temperature (23 oC). After that, a Nikon CLSM machine (C2 Plus, Nikon, Japan) was used to observe the stained sample at at two wave lengths (488 nm for living cells and 559 nm for dead cells). The biofilm thickness was measured in the 3-D scanning mode.

Additional Information

How to cite this article: Li, H. et al. Min class="Chemical">crpan>obiologically Influenced Corrosion of 2707 Hyper-Duplex Stainless Steel by Marine Pseudomonas aeruginosa Biofilm. Sci. Rep. 6, 20190; doi: 10.1038/srep20190 (2016).