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Article

Temporary Inhibition of the Corrosion of AZ31B Magnesium Alloy by Formation of Bacillus subtilis Biofilm in Artificial Seawater

by
Yaxin Kang
1,
Lei Li
2,*,
Shunling Li
1,
Xin Zhou
1,
Ke Xia
1,
Chang Liu
1 and
Qing Qu
1,*
1
School of Chemical Science and Technology, Yunnan University, Kunming 650091, China
2
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming 650091, China
*
Authors to whom correspondence should be addressed.
Materials 2019, 12(3), 523; https://doi.org/10.3390/ma12030523
Submission received: 10 December 2018 / Revised: 8 January 2019 / Accepted: 26 January 2019 / Published: 10 February 2019
(This article belongs to the Section Corrosion)
Browse Figures
Versions Notes

Abstract

:
It is well known that microorganisms tend to form biofilms on metal surfaces to accelerate/decelerate corrosion and affect their service life. Bacillus subtilis was used to produce a dense biofilm on an AZ31B magnesium alloy surface. Corrosion behavior of the alloy with the B. subtilis biofilm was evaluated in artificial seawater. The results revealed that the biofilm hampered extracellular electron transfer significantly, which resulted in a decrease of icorr and increase of Rt clearly compared to the control group. Moreover, an ennoblement of Ecorr was detected under the condition of B. subtilis biofilm covering. Significant reduction of the corrosion was observed by using the cyclic polarization method. All of these prove that the existence of the B. subtilis biofilm effectively enhances the anti-corrosion performance of the AZ31B magnesium alloy. This result may enhance the usage of bio-interfaces for temporary corrosion control. In addition, a possible corrosion inhibition mechanism of B. subtilis on AZ31B magnesium alloy was proposed.

1. Introduction

Microorganisms exhibit a strong affinity to adhere to the surface of metals [1]. This adhesion has the ability to modify the electrochemical environments of the metal/solution interface by forming a biofilm, which leads to the acceleration or deceleration of corrosion reactions [2,3]. Consequently, more and more investigation has been carried out on microbiologically influenced corrosion in recent years [4,5,6]. The corrosion behavior of Pseudomonas aeruginosa on Ni-Zn alloy and Ni-Cu alloy was explored by San et al. [7]; they showed that Pseudomonas aeruginosa accelerated Ni-Cu alloy corrosion, but inhibited Ni-Co alloy corrosion. Guo et al. [8] proved that the presence of Bacillus subtilis enhanced steel’s resistance to corrosion in seawater environment, whereas the existence of Pseudoalteromonas lipolytica worsened the alloy’s resistance to corrosion. Based on a dissertation by Duan et al. [9], single species Desulfovibrio caledoniensis or Clostridium sp. could accelerate the corrosion of carbon steel, but mixed species inhibited it. Batmanghelich et al. [10] discovered that the co-presence of Desulfovibrio vulgaris and Pseudomonas aeruginosa could inhibit cast iron corrosion more evidently, compared to a single one. As suggested above, the results are not in complete accord when diverse microorganisms or metals are used in various research conditions.
It is worth mentioning that microorganisms can influence corrosion behavior in an advantageous way, the so-called MICI (microbiologically influenced corrosion inhibition) [3,7,11]. Based on the corrosion inhibition ability of bacteria on metals, Örnek et al. [11] proposed a concept called corrosion control using regenerative biofilms. They emphasized that the inhibition ability of corrosion of bacteria should be used to provide a bioremediation method to inhibit localized corrosion of metals. Nevertheless, this research is still in a fundamental stage. This may enlighten the development of a sustainable and biological way for metal corrosion mitigation. For instance, in an oil pipeline environment, the environmental microbial community can be adjusted so that microorganisms with corrosion inhibition function as dominant strains to slow down the corrosion of metal pipelines. Thus, studies of MICI on metals will become progressively more important.
Bacillus sp., gram-positive bacteria, aerobic or facultative anaerobic, has strong tolerance to environmental changes [12]. Substantial members of Bacillus sp. have been separated out. Among them, B. subtilis is widely distributed in aquatic habitats and marine sediments [13,14,15]. Literature reports that the presence of B. subtilis could inhibit the localized corrosion of certain metals [16,17,18,19]. The corrosion behavior of B. subtilis on cold rolled steel was explored [16], which reported that the latter corrosion rate was significantly reduced in comparison with the initial rate. Örnek et al. [17] observed that the pitting corrosion of Al 2024 was visibly weakened when a B. subtilis biofilm was gradually formed. Abdoli et al. [18] showed that the bacteria of Bacillus sp. significantly enhanced the anti-corrosion performances of Al coatings. Wadood et al. [19] proved that when injected with B. subtilis, the corrosion of 304 Stainless Steel was inhibited. Based on the above literature, it is clear that the anti-corrosion properties of cold-rolled steel, aluminum, and stainless steel have been improved on formation of the B. subtilis biofilm. However, the corrosion resistance of B. subtilis biofilm to other metals has not been fully explored.
With characteristics such as good biocompatibility, low density and potential antimicrobial activity [20,21,22], magnesium alloys are extensively used in marine industries, biomedical materials, automobile manufacturing, etc. [23,24,25]. The continuous development of the light industry has increased demand for magnesium alloys, especially Mg-Al-Zn [26]. However, magnesium alloys are prone to be eroded [27,28,29,30], limiting practical applications. There are quite a few reasons that can account for the corrosion of magnesium alloys [31,32,33]; for instance, moist or polluted atmosphere, temperature, microorganisms, concentration of Cl−, Br−, SO42− and NO3−, pH, etc. Among them, the corrosion effects of microorganisms may play a significant role in analogical problems [34,35,36,37,38]. The corrosion behaviour of sulfate-reducing bacteria (SRB) on Ce-modified cast AZ91 magnesium alloy, AZ91D magnesium alloy, and 2024-T31 Al-Cu-Mg alloy were studied separately [34,35,36], which all indicated that SRB accelerated the corrosion procedure of these alloys. Besides, the corrosion behaviours of AZ31B magnesium alloy caused by the fungi of Aspergillus niger and Trichoderma harzianum were investigated. The results reflected that both fungi could decrease the resistance of corrosion on the alloy [37,38]. The above corrosion results prove that microbial biofilm can reduce anti-corrosion of magnesium alloy. There is currently no report on the improvement of magnesium alloy’s resistance to corrosion by bacterial biofilm. Therefore, the corrosion behavior of B. subtilis biofilm on AZ31B magnesium alloy was systematically researched in this work.

2. Materials and Methods

2.1. Bacterium and Medium

B. subtilis was supported by the State Key Laboratory for Conservation and Utilization of Bio-resources in Yunnan (Kunming, China). B. subtilis was incubated in Luria-Bertani medium (LB). Optical density (OD550) of the broth was measured after dilution, with a value of 0.7. Number of colony forming units (CFU) was about 1 × 107 CFU mL−1 for the following experiments.
The study was carried out with artificial seawater medium. The composition is as follows [16], NaCl: 17.6 g L−1, MgCl2·6H2O: 1.87 g L−1, Na2SO4: 1.47 g L−1, CaCl2·2H2O: 0.41 g L−1, KCl: 0.25 g L−1, NaHCO3: 0.08 g L−1, KBr: 0.04 g L−1, Na2HPO4: 0.01 g L−1, FeSO4·7H2O: 0.01 g L−1, SrCl2·6H2O: 0.008 g L−1, H3BO3: 0.008 g L−1, tryptone: 2.0 g L−1, starch: 1.0 g L−1, yeast extract: 1.0 g L−1, pH: 7.5 ± 0.1.

2.2. Material and Specimen

AZ31B magnesium alloy, 5 mm thick sheets. Each of the alloy coupon was cut to a size of 20 mm × 20 mm. Table 1 displays the chemical compositions of the AZ31B magnesium alloy.
For the electrochemical measurement, magnesium alloy specimens were made into a working electrode. The alloy samples were connected with copper wire of 20 cm and fixed with epoxy resin glue. The exposed area of the working electrodes in artificial seawater medium was 20 × 20 mm2. In the surface topography test, the soaking volume of magnesium alloy samples in artificial seawater medium was 20 × 20 × 5 mm3. All the alloy samples were polished sequentially with SiC papers (320–1200 grit), then cleaned with distilled water in an ultrasonic cleaner for 5 min. Under irradiation of an ultraviolet lamp, the samples were disinfected with 75% ethanol and 2.5% glutaraldehyde for 1 h, respectively.

2.3. Scanning Electron Microscopy/Energy Dispersive X-ray Spectra Analysis (SEM & EDS)

For SEM & EDS test, the specimens were submerged in 2.5% glutaraldehyde for 15 min, and washed with distilled water. The samples were then dehydrated with different concentrations of alcohol. The type of instrument used for the test is Philips XL30 ESEM-TMP, Amsterdam, Netherlands.

2.4. Electrochemical Techniques

2.4.1. Electrochemical Corrosion

To characterize the corrosion behaviour of the samples, electrochemical techniques (cyclic polarization, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), open circuit potential (OCP)), were carried out by means of an electrochemical workstation from Perkin Elmer Company (PARSTAT 2263, Waltham, MA, USA). A prescriptive three-electrode system was selected, with a platinum plate as the counter electrode, the AZ31B magnesium alloy specimens as the working electrodes, and a saturated calomel electrode (SCE) as the reference electrode.
OCP was conducted for 1000 s with 5 s intervals after stabilization for 10 min at each testing time (12, 24, 48, 96 h); the stable value of Eocp at each testing time was then recorded. EIS measurement was performed by a sinusoidal wave with 10 mV amplitude in the frequency range of 10−1 to 105 Hz. In the potentiodynamic polarization test, the potential range was −0.25 V to +0.50 V vs. SCE, and scan rate was 0.5 mV s−1. Cyclic polarization measurement was conducted with a potential range of −0.50 V to +0.50 V vs. SCE, at a scan rate of 0.5 mV s−1.

2.4.2. Electron Transfer Property

Electron transfer capacity of B. subtilis was measured using a CHI660 electrochemical tester (Chenhua, Shanghai, China). Here, a saturated Ag/AgCl electrode was used as the reference electrode, a platinum wire was used as the counter electrode, and a glassy carbon disk (Ø = 3 mm) was used as the working electrode. The working electrode was polished with alumina powder of 0.3 μm and 0.05 μm. After that, the working electrode was cleaned by ultrasonic with pure water. In the cyclic voltammetry (CV) test, the range of potential was from −0.95 V to +0.10 V vs. Ag/AgCl, and the scan rate was 10 mV s−1. In the differential pulse voltammetry (DPV) test, the potential range was from −0.76 V to −0.50 V vs. Ag/AgCl, sampling width was 16.7 ms, potential increment was 4 mV, quiet time was 2 s, pulse width was 50 ms, pulse amplitude was 50 mV, and sensitivity was 10−6 A V−1.

2.5. Ultraviolet-Visible Spectroscopy Analysis (UV-VIS)

In UV-VIS experiments, the corrosion products or B. subtilis metabolites were kept separate, and analyzed them by ultraviolet and visible spectroscopy (UV-2401PC, Kyoto, Japan).

2.6. Fourier Transform Infrared Spectroscopy Analysis (FTIR)

For this test, the corrosion products or B. subtilis metabolites were separated, and then tested. These samples were carried out by KBr tableting technique. The type of instrument used for the test is Thermo Fisher Scientific Nicolet IS10, Waltham, MA, USA.

3. Results

3.1. SEM & EDS

Figure 1 displays the surface topography of AZ31B magnesium alloy specimens in control group (A) and B. subtilis presence group (B) at 48 h. In the control group (Figure 1A), the specimen surface is matt, and mainly has massive and deep large cracks. The largest crack on the specimen’s surface of control group is about 4.30 μm in size. In the B. subtilis presence group (Figure 1B), abundant elliptical particles are spread over on AZ31B magnesium alloy surface. In addition, a dense and continuous biofilm of B. subtilis was formed on the AZ31B magnesium alloy surface.
The composition of elements on the surface of specimens is explored by EDS; the test position is the red square in Figure 1A,B. The major elements on the coupons’ surface are presented in Table 2. Compared with the control group, the EDS results of the B. subtilis presence group indicate that the content of C, O, P elements has increased. These are key elements of extracellular polymeric substances (EPS) on microbes. EPS consists of proteins, nucleic acids, polysaccharides, lipids, enzymes, etc. [39]. The EDS result demonstrates that a biofilm of B. subtilis has been formed on the alloy specimen surface. After electrochemical measurement, the surface corrosion of the alloy is slightly aggravated, but the basic morphology and elemental composition has not changed significantly, as shown in Figure S1 and Table S1 of Supplementary Materials.

3.2. Electrochemical Corrosion

3.2.1. OCP

The OCP changes with the immersion time of AZ31B magnesium alloy electrodes in the control group and B. subtilis presence group (shown in Figure 2). The changing trend of open circuit potential (Eocp) over time in the B. subtilis presence group and control group is in accordance: they all move negatively first, then shift positively, and finally move negatively. However, OCP curves reveal that Eocp of the B. subtilis presence group is more positive than that of the control group. Some researchers also observed an increase in OCP of different metals with microbes, compared to the sterile ones. Örnek et al. [3,11,17] proposed that an ennoblement of Eocp was detected when B. subtilis biofilm was present; the biofilm was able to improve the corrosion resistance of aluminum 2024 and brass. Mansfeld [2] pointed out that Shewanella oneidensis and algae always increased the corrosion rate of metals with a more negative shift of Eocp, but B. subtilis could reduce the corrosion rate with a more positive shift of Eocp. In these researches, the ennoblement of Eocp with B. subtilis, compared to the sterile solution, demonstrates that the presence of B. subtilis effectively enhances the anti-corrosion performance of AZ31B magnesium alloy.

3.2.2. Potentiodynamic Polarization

The potentiodynamic polarization variations with immersion time of AZ31B magnesium alloy coupons in control group and B. subtilis presence group are shown in Figure 3. The electrochemical parameters are calculated and given in Table 3. Depending on Figure 3, compared with the control group, corrosion potential (Ecorr) of the B. subtilis presence group increased gradually, and corrosion current density (icorr) significantly reduced with the prolongation of soaking time, which displays that the presence of B. subtilis improves the anti-corrosion performance of AZ31B magnesium alloy. Additionally, Table 3 shows that icorr of the control group significantly increased in the early stage, and then slightly decreased. The parameters’ variation of the control group can be because the intrinsic layer (Mg or Al) of the alloy surface is dissolved in the early immersion stage—icorr increased progressively. The corrosion products are then amassed gradually on the alloy surface, which form a product film like Mg(OH)2, Al(OH)3, etc. [40]. The product film hinders contact between the artificial seawater medium and the alloy interface, and causes a slight decline in icorr. In the B. subtilis presence group, icorr is clearly decreased and then slightly increased with soaking time. The probable reason is that a compact layer of B. subtilis biofilm is formed gradually in the early stage of soaking, which clearly hinders the corrosion of the AZ31B magnesium alloy. Accordingly, icorr decreases significantly. However, after 48 h of immersion, the biofilm layer gradually exfoliates due to the consumption of nutrients; icorr was found to have increased slightly.
To sum up, Ecorr of the B. subtilis presence group increased clearly, and icorr reduced significantly, when compared with the control group. This shows that the presence of B. subtilis can effectively inhibit corrosion of the AZ31B magnesium alloy.

3.2.3. Cyclic Polarization

Cyclic polarization is a relatively nondestructive method to evaluate the corrosion susceptibility of materials [41]. Figure 4 displays cyclic polarization variations with the immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. Figure 4 shows that all curves appear to be hysteresis loops, protection potential (Ep). Interestingly, the relative area of hysteresis loop in the B. subtilis presence group reduced significantly, when compared to the control group. The Ep in the B. subtilis presence group is steadily higher than that in the control group. The Ep values of the B. subtilis presence group: −1524.3 mV (12 h), −1481.2 mV (24 h), −1431.1 mV (48 h), −1479.4 mV (6 h); the Ep values of the control group: −1492.2 mV (12 h), −543.6 mV (24 h), −1535.0 mV (48 h), −1565.9 mV (96 h). These suggest that corrosion of the AZ31B magnesium alloy is effectively inhibited when it is exposed to the B. subtilis environment [41]. In the B. subtilis group, Ep reaches a maximum at 48 h, which may be caused by the formation of a compact B. subtilis biofilm on the AZ31B magnesium alloy surface. This biofilm hinders contact between the artificial seawater and the alloy surface. The cyclic polarization curves prove that the existence of B. subtilis biofilm enhances the anti-corrosion performance of the AZ31B magnesium alloy.

3.2.4. EIS

The corrosion characteristics of AZ31B magnesium samples were performed by EIS after exposure to artificial seawater or inoculation with and without B. subtilis (plotted in Figure 5). Figure 5A–D is Nyquist diagrams, Figure 5A’–D’ is Bode modulus diagrams and Bode phase angle diagrams, respectively. All the Nyquist diagrams in Figure 5A–D show analogous and incomplete semicircles. The magnitude of the semicircle diameter reflects the degree of electron transfer. If the diameter is large, it means the electron transfer process is greatly hindered; the electron transfer rate is slow, and displays a small amount of electronic exchange in unit time. In other words, the coupon is corroded difficultly, which appears as corrosion inhibition [42,43,44,45]. Figure 5A–D reflect that the anti-corrosion performance of AZ31B magnesium alloy samples in the B. subtilis presence group is increased significantly with immersion time, when compared to the control group, which presumably is attributed to the formation of the B. subtilis biofilm. That is, B. subtilis biofilm can slightly improve the anti-corrosion characteristics of AZ31B magnesium alloy. An inductive impedance arc appears in a low frequency range in the B. subtilis presence group, especially after immersion for 48 h, which is put down to the formation of corrosion source on the AZ31B magnesium alloy surface in the corrosion induction period. In this period, the thickness of B. subtilis biofilm is a stated variable; the Faraday current decreased with an increase in thickness of the B. subtilis biofilm [46]. Figure 5A–D shows that corrosion resistance is the strongest at 48 h; hence, the thickness of the B. subtilis biofilm is the highest at that time. In the control group, the impedance value is decreased significantly and then slightly increased. It is mainly because the magnesium alloy is directly exposed in the artificial seawater medium, the surface is dissolved, and the corrosion rate is gradually increased. However, with time, a corrosion product is gradually formed and covers the alloy surface, which restricts the rapid transmission of erosive ions and molecules to the matrix materials [40]. This leads to an increase of the impedance value of the latter. In the B. subtilis presence group, the impedance value is increased firstly and then decreased. It is caused mainly by the formation of the B. subtilis biofilm on the AZ31B magnesium alloy surface with immersion time, leading to a clear reduction in corrosion rate. However, after soaking for 48 h, the biofilm layer is gradually exfoliated and the nourishing substances are depleted. Therefore, the impedance value decreases.
The corresponding numerical values of the electrochemical parameters modeled by the equivalent electrical circuit are shown in Table 4. The corrosion inhibition efficiency (η) can be obtained by the formula η = (Rt’ − Rt)/Rt’ × 100%. It can be calculated as: η is 14.7% (12 h), 68.8% (24 h), 91.4% (48 h), and 69.4% (96 h), respectively. These prove that the B. subtilis biofilm can effectively increase the anti-corrosion performance of the AZ31B magnesium alloy. Moreover, at 48 h, corrosion inhibition is the strongest.

3.3. Electron Transfer Property

CV and DPV measurements are shown in Figure 6. Figure 6A presents two CV curves. In the control group, the CV curve shows a weak oxidation peak in the range of −0.7 V to −0.6 V. However, the oxidation peak of the B. subtilis presence group is slight. It is almost invisible, when compared to the control group, which suggests that the electronic transmission capacity is weakened by B. subtilis. Figure 6B displays two DPV curves. The oxidation peak of the B. subtilis presence group is at −0.635 V, the oxidation peak of control group is at −0.645 V, and the current signals in the absence and presence of B. subtilis are 12.9 µA cm−2 and 11.8 µA cm−2, respectively. That is, the current signal of the B. subtilis presence group decreases, compared to the control group, which suggests that the B. subtilis biofilm inhibits electronic transmission from the electron donor to the electron receptor [47,48,49]. The causation of alterations of CV and DPV in the B. subtilis presence group is ascribed to the adhesion of B. subtilis cells and EPS on the working electrode surface. These tangibly demonstrate that B. subtilis plays an inhibitory role in the electron transfer process.

3.4. UV-VIS

Figure 7 shows the UV-VIS spectra of B. subtilis, AZ31B magnesium alloy inoculation with or without B. subtilis after immersion in artificial seawater for 48 h. In the spectrogram of B. subtilis, there is a weak absorption peak at 336 nm, which may be caused by the n-π* transition of aldehydes or ketones in the metabolites of B. subtilis. The spectrogram of the AZ31B magnesium alloy in the presence of B. subtilis is clearly different in the spectrograms of B. subtilis and AZ31B magnesium alloy. It has a relatively conspicuous absorption peak at 259 nm, compared with other groups. The absorption peak may be generated by the π-π* transition; it can be supposed that the absorption peak is caused by the interaction between the metabolites of B. subtilis and the AZ31B magnesium alloy.

3.5. FTIR

Figure 8 shows the spectrum graph of the AZ31B magnesium alloy; the absorption peaks of 3689 cm−1, 3401 cm−1, 1440 cm−1 represent an O–H bond and the sharp peak at 534 cm−1 is assigned to the Mg–O bond [50]. In the spectrum graph of B. subtilis, the peak of 3200–3500 cm−1, 1639 cm−1, 1095 cm−1, 675 cm−1 represent the O–H, C–O, C=O bonds of polysaccharides in the metabolic products of B. subtilis [51,52,53,54,55,56]. The peaks at 3200–3500 cm−1, 1424 cm−1 represent N–H, C–N bonds from proteins in the metabolic products of B. subtilis [53,54]. When comparing the spectrum graph of the AZ31B magnesium alloy in the presence of B. subtilis with the other two graphs, the main difference is that the characteristic peaks at 773 cm−1 and 534 cm−1, 416 cm−1 are caused by the Al–O, Mg–O bond from complexes of metal ion-organic ligand [57]. This means that a dense B. subtilis biofilm is formed on the alloy surface, which consists of protein, polysaccharides, etc. Sunde et al. [58] reported that macromolecular hydroxyl compounds have a certain affinity with metal cations and form metal-organic ligand compounds, which facilitate the strong binding of EPS and AZ31B magnesium alloy.

4. Discussion

According to the SEM & EDS tests (Figure 1), potentiodynamic polarization (Figure 3), cyclic polarization (Figure 4) and EIS (Figure 5), significant corrosion inhibition occurs when AZ31B magnesium alloy is in artificial seawater medium with a B. subtilis biofilm. Outcome of the electron transfer property (Figure 6) indicates that the presence of B. subtilis hinders the transfer of electrons. In addition, the investigations of UV-VIS (Figure 7) and FTIR (Figure 8) prove that metal ion-organic ligand complexes are formed on the alloy surface. These complexes can firmly bind EPS to the AZ31B magnesium alloy. From the experiments, we put forward a possible mechanism for the presence and absence of B. subtilis.
In the control group, when the alloy is immersed in artificial seawater solution, the surface metals of Mg and Al are dissolved to form corrosion products. These metals in active sites are relatively easier to dissolve, so corrosion occurs through cracks. Anodic dissolution of the magnesium alloy is balanced by the cathodic reduction reaction:
Mg → Mg2+ + 2 e−
Al → Al3+ + 3 e−
H2O + 1/2 O2 + 2e− → 2 OH−
In the B. subtilis presence group, icorr exhibits significant decrease compared to the control group. This indicates that the corrosion resistance of AZ31B magnesium alloy is improved. Örnek et al. [17] observed when B. subtilis secreted polyaspartic acid or polyglutamic acid, it inhibited corrosion. However, components and concentrations of microbial secretions are affected by mediums with different nutrients. Thus, this mechanism type of inhibition-secretion is not suitable for this study. Based on a review by Xu et al. [1], corrosion mechanism could be divided into aerobic corrosion and anaerobic corrosion. When B. subtilis adheres firmly to the surface of the AZ31B magnesium alloy to form a dense biofilm, it is mainly anaerobic corrosion. This dense biofilm hinders the transfer of electrons from the anode to the cathode, which makes corrosion reaction long and tedious, leading to gradual corrosion inhibition of the magnesium alloy [1]. Liu et al. [8] proved that when compared to loose biofilm, the existence of dense and compact B. subtilis biofilm enhanced metallic resistance to corrosion. Therefore, an anti-corrosion mechanism of B. subtilis biofilm on the alloy surface is shown in Figure 9. In the early stages of immersion, microbes of B. subtilis are at a reproductive stage and the alloy surface does not have a biofilm covering. The planktonics of B. subtilis cells are preferred for adherence to the alloy surface through electrostatic attraction force (Figure 9B) [59]. Moreover, B. subtilis cells metabolize and propagate on the alloy surface, and form a thin biofilm (Figure 9C) [60]. The metabolites of B. subtilis then combine with magnesium ions of the alloy surface to form metal ion-organic ligand complexes, which make the biofilm denser and firmer (Figure 9D). The dense biofilm of B. subtilis does not only delay the dissolution of the magnesium alloy, but also hinders electron transfer and diffusion, which results in a decrease of icorr. The anti-corrosion performance of the alloy is thus enhanced.

5. Conclusions

Electrochemical techniques and surface topography analysis methods were used to analyze the corrosion inhibition behaviour of B. subtilis biofilm on a AZ31B magnesium alloy surface in artificial seawater. The following conclusions were obtained:
1. B. subtilis can form a compact biofilm on the AZ31B magnesium alloy surface.
2. Potentiodynamic polarization, EIS, cyclic polarization, and SEM results significantly inhibit corrosion when a B. subtilis biofilm is present on the AZ31B magnesium alloy surface.
3. The investigation results of UV-VIS, FTIR, CV and DPV testify that complexes such as Al3+-organic acid and Mg2+-organic acid are formed on the alloy surface. These complexes firmly attach the biofilm to the alloy surface. The biofilm effectively hinders the transfer of electrons and safeguards the alloy.

Supplementary Materials

The following are available online at https://www.mdpi.com/1996-1944/12/3/523/s1, Figure S1. SEM images of AZ31B magnesium alloy specimens before and after electrochemical measurement in the control group (before: A, after: B) and the B. subtilis presence group (before: C, after: D) at 48 h. (high voltage: 20 kV). Table S1. Elemental compositions (wt %) of the surface of AZ31B magnesium alloy specimens before and after electrochemical measurement in the control group and the B. subtilis presence group.

Author Contributions

Q.Q. and L.L. proposed and guided the project. Y.K. prepared the figures and wrote the paper. S.L., X.Z., K.X. and C.L. revised the manuscript. All authors joined the discussion and made useful suggestions.

Funding

This investigation is financed by the National Natural Science Foundation of China under Grant Nos. 51661033, 51361028, 51161025, and 31660538.

Conflicts of Interest

The authors declare no conflict of interest.

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Materials 12 00523 g001 550
Figure 1. SEM images of AZ31B magnesium alloy specimens in control group (A) and B. subtilis presence group (B) at 48 h. (high voltage: 20 kV).
Figure 1. SEM images of AZ31B magnesium alloy specimens in control group (A) and B. subtilis presence group (B) at 48 h. (high voltage: 20 kV).
Materials 12 00523 g001
Materials 12 00523 g002 550
Figure 2. The changes of Eocp with immersion time of AZ31B magnesium alloy specimens in control group and B. subtilis presence group. Scatter bands represent the standard deviations of the three tests.
Figure 2. The changes of Eocp with immersion time of AZ31B magnesium alloy specimens in control group and B. subtilis presence group. Scatter bands represent the standard deviations of the three tests.
Materials 12 00523 g002
Materials 12 00523 g003 550
Figure 3. Potentiodynamic polarization curves with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. (A) 12 h; (B) 24 h; (C) 48 h; (D) 96 h.
Figure 3. Potentiodynamic polarization curves with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. (A) 12 h; (B) 24 h; (C) 48 h; (D) 96 h.
Materials 12 00523 g003
Materials 12 00523 g004 550
Figure 4. Cyclic polarization curves with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. (A) 12 h; (B) 24 h; (C) 48 h; (D) 96 h.
Figure 4. Cyclic polarization curves with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. (A) 12 h; (B) 24 h; (C) 48 h; (D) 96 h.
Materials 12 00523 g004
Materials 12 00523 g005a 550Materials 12 00523 g005b 550
Figure 5. EIS with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. (A–D): Nyquist diagrams, (A’–D’): the Bode modulus diagrams and Bode phase angle diagrams.
Figure 5. EIS with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. (A–D): Nyquist diagrams, (A’–D’): the Bode modulus diagrams and Bode phase angle diagrams.
Materials 12 00523 g005aMaterials 12 00523 g005b
Materials 12 00523 g006 550
Figure 6. CV curves (A) of the control group and B. subtilis presence group at 48 h, DPV curves (B) of the control group and B. subtilis presence group at 48 h.
Figure 6. CV curves (A) of the control group and B. subtilis presence group at 48 h, DPV curves (B) of the control group and B. subtilis presence group at 48 h.
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Materials 12 00523 g007 550
Figure 7. UV-VIS analysis of the AZ31B magnesium alloy, B. subtilis, and AZ31B magnesium alloy with inoculation of B. subtilis at 48 h.
Figure 7. UV-VIS analysis of the AZ31B magnesium alloy, B. subtilis, and AZ31B magnesium alloy with inoculation of B. subtilis at 48 h.
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Materials 12 00523 g008 550
Figure 8. FTIR spectra of AZ31B magnesium alloy, B. subtilis, and AZ31B magnesium alloy with inoculation of B. subtilis at 48 h.
Figure 8. FTIR spectra of AZ31B magnesium alloy, B. subtilis, and AZ31B magnesium alloy with inoculation of B. subtilis at 48 h.
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Materials 12 00523 g009 550
Figure 9. Corrosion effect of B. subtilis biofilm on the AZ31B magnesium alloy surface. (A) Substrate material; (B) Cells adhesion; (C) Thin biofilm; (D) Dense biofilm.
Figure 9. Corrosion effect of B. subtilis biofilm on the AZ31B magnesium alloy surface. (A) Substrate material; (B) Cells adhesion; (C) Thin biofilm; (D) Dense biofilm.
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Table
Table 1. Chemical compositions of AZ31B magnesium alloy (wt %).
Table 1. Chemical compositions of AZ31B magnesium alloy (wt %).
ElementAlZnMnSiNiFeCuMg
wt %3.050.990.280.0250.00490.0030.002balance
Table
Table 2. Elemental compositions (wt %) of the surface of AZ31B magnesium alloy specimens in the control group and B. subtilis presence group.
Table 2. Elemental compositions (wt %) of the surface of AZ31B magnesium alloy specimens in the control group and B. subtilis presence group.
Element (wt %)CONaMgAlPSClCaOthers
The sterile control group0.8123.630.5562.523.480.670.370.327.65<0.001
The B. subtilis presence group6.1133.392.3531.614.0611.900.572.407.61<0.001
Table
Table 3. Potentiodynamic polarization parameters with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group.
Table 3. Potentiodynamic polarization parameters with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group.
GroupsTime
(h)		Ecorr
(mV) vs. SCE		icorr
(μA cm−2)		βa
(mV deg−1)		βc
(mV deg−1)
The sterile control group12−1415.4 ± 0.183.0 ± 0.5152.5 ± 9.2−113.0 ± 3.6
24−1410.7 ± 0.1166.4 ± 0.7193.2 ± 6.8−89.8 ± 7.4
48−1427.7 ± 0.1243.3 ± 1.2203.7 ± 10.5−52.1 ± 10.2
96−1423.1 ± 0.2116.4 ± 0.8199.7 ± 11.6−37.1 ± 6.3
The B. subtilis presence group12−1362.4 ± 0.147.4 ± 0.6209.7 ± 7.2−48.1 ± 5.4
24−1341.1 ± 0.218.3 ± 1.0189.5 ± 9.3−52.3 ± 6.8
48−1356.9 ± 0.114.5 ± 0.9117.2 ± 9.8−151.3 ± 11.3
96−1291.3 ± 0.116.3 ± 0.9113.1 ± 10.2−72.1 ± 8.9
Table
Table 4. EIS parameters with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. Rs represents the solution resistance, Cdl represents double layer capacitance, Rt represents charge-transfer resistance, L represents equivalent inductance, RL represents equivalent resistance.
Table 4. EIS parameters with immersion time of AZ31B magnesium alloy coupons in the control group and the B. subtilis presence group. Rs represents the solution resistance, Cdl represents double layer capacitance, Rt represents charge-transfer resistance, L represents equivalent inductance, RL represents equivalent resistance.
GroupsTime
(h)		Rs
(Ω cm2)		Cdl
(×10−5 F cm−2)		Rt
(Ω cm2)		L
(H cm2)		RL
(Ω cm2)
The sterile control group126.5 ± 0.14.1 ± 0.176.9 ± 1.839.1 ± 1.753.2 ± 0.2
247.0 ± 0.28.5 ± 0.340.9 ± 0.818.4 ± 0.915.2 ± 0.2
485.9 ± 0.12.2 ± 0.313.8 ± 2.21.8 ± 2.58.1 ± 0.23
963.8 ± 0.39.3 ± 0.336.8 ± 0.59.23 ± 0.747.4 ± 0.1
The B. subtilis presence group127.7 ± 0.27.7 ± 0.290.2 ± 0.740.9 ± 0.981.7 ± 0.2
2416.0 ± 0.23.5 ± 0.2131.1 ± 0.991.6 ± 1.267.2 ± 0.2
486.5 ± 0.29.8 ± 0.5161.4 ± 1.298.2 ± 2.4132.8 ± 0.3
968.3 ± 0.28.1 ± 0.1120.3 ± 0.363.1 ± 0.8493.3 ± 0.2

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Kang, Y.; Li, L.; Li, S.; Zhou, X.; Xia, K.; Liu, C.; Qu, Q. Temporary Inhibition of the Corrosion of AZ31B Magnesium Alloy by Formation of Bacillus subtilis Biofilm in Artificial Seawater. Materials 2019, 12, 523. https://doi.org/10.3390/ma12030523

AMA Style

Kang Y, Li L, Li S, Zhou X, Xia K, Liu C, Qu Q. Temporary Inhibition of the Corrosion of AZ31B Magnesium Alloy by Formation of Bacillus subtilis Biofilm in Artificial Seawater. Materials. 2019; 12(3):523. https://doi.org/10.3390/ma12030523

Chicago/Turabian Style

Kang, Yaxin, Lei Li, Shunling Li, Xin Zhou, Ke Xia, Chang Liu, and Qing Qu. 2019. "Temporary Inhibition of the Corrosion of AZ31B Magnesium Alloy by Formation of Bacillus subtilis Biofilm in Artificial Seawater" Materials 12, no. 3: 523. https://doi.org/10.3390/ma12030523

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