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Enhancement of exogenous riboflavin on microbiologically influenced corrosion of nickel by electroactive Desulfovibrio vulgaris biofilm

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Cell counts and biofilm observation

Figure 1a–e show the schematic representation of the hemocytometer and optical images of D. vulgaris cells in the counting chamber obtained using an optical microscope (XSP-BM-2CA, BM Optical Instruments Manufacture Co., Ltd, Shanghai, China). The D. vulgaris cells were mobile under the optical microscope, making it easy to identify D. vulgaris cells from black sulfides. Figure 1 also shows sessile cell counts on Cu and Ni coupon surfaces without and with riboflavin, which were (1.9 ± 0.2) × 108 cells cm−2, (1.8 ± 0. 4) × 108 cells cm−2, (1.9 ± 0. 3) × 108 cells cm−2, and (2.0 ± 0. 2) × 108 cells cm−2, respectively. The growth curves of D. vulgaris planktonic cells (Supplementary Fig. 2, supporting information) in Cu and Ni vials without and with riboflavin were quite close during the 7-d incubation. The cell count results indicated that the addition of riboflavin did not significantly change the abundance of the sessile cells for the two metals.

Fig. 1: Optical micrographs of D. vulgaris and sessile D. vulgaris cell counts.
figure 1

Schematic diagram of counting chamber (ae) correspond to sessile D. vulgaris on the blood cell counting plate (motile D. vulgaris underlined as white spots in red circles), and sessile D. vulgaris counts after the 7-d incubation: b Cu + D. vulgaris only; c Cu + D. vulgaris + riboflavin; d Ni + D. vulgaris only; e Ni + D. vulgaris + riboflavin. (Error bars stand for the standard deviations from at least three independent coupons).

Figure 2 displays the fluorescent microscope (FM) (Axio Scope A1, Carl Zeiss, Jena, Germany) images and field emission scanning electron microscopy (FE-SEM) (Gemini 300, Carl Zeiss, Jena, Germany) images of D. vulgaris biofilm formed on Cu and Ni coupons after the 7-d incubation in D. vulgaris media without and with riboflavin. After staining, the green and red dots in FM images (Fig. 2a–d) represent the live and dead D. vulgaris cells, respectively. After the 7-d incubation, the live and only a few dead sessile D. vulgaris cells were discovered on two metal coupons, indicating that D. vulgaris biofilms were in health. The FE-SEM images (Fig. 2a’–d’) show that Ni surfaces (Fig. 2c’ and d’) have more sessile D. vulgaris cells than Cu (Fig. 2a’ and b’), which is consistent with the sessile cell counts (Fig. 1).

Fig. 2: D. vulgaris biofilm morphology.
figure 2

FM and SEM images of biofilms on Cu and Ni coupons after the 7-d incubation in the D. vulgaris media: a, a‘ Cu + D. vulgaris only; b, b‘ Cu + D. vulgaris + riboflavin; c, c‘ Ni + D. vulgaris only; d, d‘ Ni + D. vulgaris + riboflavin.

Gas concentration in the headspace

Figure 3 shows the headspace H2 concentration of the 75 mL headspace in anaerobic vials (Cu + D. vulgaris, Ni + D. vulgaris, Cu + Abiotic, Ni + Abiotic, and D. vulgaris only) during the 7-d incubation. The abiotic headspace H2 curves in Fig. 3 varied linearly during the 7-d incubation period, and the H2 concentration in the vial with Cu and Ni reached 50 ± 5 ppm (v/v), 60 ± 8 ppm (v/v), respectively. The biotic H2 evolution curves were all inverted V shape. The H2 evolution curve in D. vulgaris vial containing Ni coupons was very similar to that of D. vulgaris media alone, with a peak H2 concentration occurring on the 3rd day, about (7.0 ± 0.5) × 102 ppm (v/v). In contrast, a significant rise in H2 concentration was detected in D. vulgaris vial containing Cu coupons, with a peak headspace H2 concentration of (2.1 ± 0.2) × 103 ppm (v/v).

Fig. 3: Headspace H2 concentration.
figure 3

Headspace H2 concentration of the 75 mL headspace in anaerobic vials (Cu + D. vulgaris, Ni + D. vulgaris, Cu + Abiotic, Ni + Abiotic, and D. vulgaris only) during the 7-d incubation (Error bars stand for the standard deviations from at least three independent coupons).

Surface corrosion product analysis

Figure 4 shows the X-ray diffraction (XRD) (D8 advance, Bruker, Karlsruhe, Germany) patterns of corrosion product films on Cu and Ni coupons after the 7-d incubation in D. vulgaris media without and with riboflavin. The corrosion products of Cu were Cu2S. However, the Ni XRD patterns only identified the typical Ni peaks, without traces of Ni compounds, indicating that Ni compounds probably existed in the film in the amorphous form.

Fig. 4: XRD.
figure 4

XRD patterns of the corrosion products on Cu and Ni coupons after the 7-d incubation in the D. vulgaris media without and with riboflavin.

To study the compositions of corrosion products, X-ray photoelectron spectroscopy (XPS) (Kratos Axis UltraDLD, Shimadzu, Kyoto, Japan) measurements were conducted on Cu and Ni coupons after the 7-d incubation in D. vulgaris media without and with riboflavin, as displayed in Fig. 5. The XPS spectral lines of Cu 2p, Ni 2p, and S 2p were fitted by Gaussian fitting and the corresponding binding energy (BE) are listed in Supplementary Table 1 (supporting information). As shown in Fig. 5a and b, the Cu 2p1/2 peak was located at 952.4 eV, the Cu 2p3/2 peak was located at 932.4 eV, and the S 2p3/2 peak was located at 161.5 eV, corresponding to the BE of Cu+ and S2+ in the standard data of Cu2S. In Fig. 5c, the two typical peaks with BE of 874.4 and 856.4 eV were ascribed to Ni 2p1/2 and Ni 2p3/2, respectively, which were characteristic of NiS. In addition, there were also two satellite peaks at 861.5 and 879.8 eV (Fig. 5c). The two peaks at 162.7 eV (S 2p1/2) and 161.5 eV (S 2p1/2) were ascribed to the presence of NiS in Fig. 5d.

Fig. 5: XPS.
figure 5

XPS spectra of corrosion products on the surface of Cu and Ni coupons after the 7-d incubation in the D. vulgaris media without (I) and with (II) riboflavin: a Cu 2p, b S 2p, c Ni 2p, d S 2p.

Weight loss and pit analysis

The abiotic weight loss for Cu and Ni coupons after the 7-d immersion in the sterile medium with riboflavin was within the range of weighing error (Fig. 6a). Supplementary Fig. 3 (supporting information) shows the surface profile curves of corrosion pits on Cu and Ni coupons after the 7-d immersion in the sterile medium without and with riboflavin. There was no statistically significant difference among the four pit depth data. 20 ppm (w/w) riboflavin did not cause or accelerate the corrosion of Cu and Ni coupons in the sterile medium. Figure 6a also shows the biotic weight loss data of Cu and Ni coupons after the 7-d incubation in D. vulgaris media without and with riboflavin. There were 26.6 ± 3.1, 27.5 ± 2.6, 1.2 ± 0.2, and 1.9 ± 0.3 mg cm−2 corresponding to Cu without riboflavin, Cu with riboflavin, Ni without riboflavin, and Ni with riboflavin, respectively. In the presence of D. vulgaris, the addition of riboflavin resulted in a 59% increase in weight loss after 7-d incubation of corrosion in Ni, whereas the weight loss increase of Cu was insignificant.

Fig. 6: Weight losses and pit depth.
figure 6

a Weight losses and b pit depth of Cu and Ni coupons after the 7-d incubation in the D. vulgaris media without and with riboflavin. (Error bars stand for the standard deviations from at least three independent coupon).

The confocal laser scanning microscope (CLSM) (VK-X250 K, Keyence, Osaka, Japan) was used to examine the whole coupon surfaces at low magnification to identify the area with the deepest pit, and then obtain the maximum pit depth data (Figs. 6b, 7) at high magnification. After the addition of riboflavin, the maximum pit depth of Cu increase was statistically insignificant (22.1 ± 5.9 vs. 25.2 ± 2.5 μm), whereas the maximum pit depth of Ni was from 5.4 ± 0.8 to 13.0 ± 2.1 μm, increased by 140%. Unlike Ni MIC, Cu corrosion was severe uniform corrosion accompanied by pitting, and the original polished lines on Cu surfaces have disappeared (Fig. 7a, b). In fact, pitting is much more serious than uniform corrosion, because surface polishing lines are still visible (Fig. 7c, d), which makes pitting difficult to detect.

Fig. 7: CLSM.
figure 7

The typical pitting 3D morphologies and depth of the Cu and Ni coupons after the 7-d incubation in the D. vulgaris media: a Cu + D. vulgaris only; b Cu + D. vulgaris + riboflavin; c Ni + D. vulgaris only; d Ni + D. vulgaris + riboflavin.

Cyclic voltammetry of D. vulgaris biofilms

The steady-state cyclic voltammetry (CV) test was performed in a pH 7.2 phosphate buffered saline (PBS) solution to assess the electroactivity of the D. vulgaris biofilm formed on graphite electrodes (GEs), as shown in Fig. 8. GEs were taken from the D. vulgaris media and evaluated in sterile de-oxygenated PBS to exclude the effect of metabolites. Figure 8a and b show CV curves by various scan rates after the 7-d incubation in D. vulgaris media without and with riboflavin, respectively. The obvious redox peak current signal (red line) was observed in Fig. 8c, suggesting that the electroactivity of the D. vulgaris biofilm was enhanced by adding riboflavin. At 100 mV s−1 scan rate, the redox peak current increased approximately threefold after adding riboflavin, indicating the D. vulgaris biofilm was positively correlated with soluble riboflavin. In addition, the relationship between the current density difference and the scanning rate (Fig. 8d), i.e., the linear slope value, is used to indicate the electroactivity of D. vulgaris biofilms. A steeper slope denotes a more electroactive biofilm.

Fig. 8: CV.
figure 8

CV of biofilm in the PBS after the 7-d incubation in the D. vulgaris media without (a) and with (b) riboflavin by various scan rates, c comparison of CV curve without (black) or with (red) the riboflavin by 100 mV s−1 scan rate, d current density differences plotted against scan rates.

Electrochemical measurements

Figure 9 shows the electrochemical results of Cu and Ni coupons after the 7-d immersion in the sterile medium without and with riboflavin. The abiotic impedance of Cu and Ni coupons slightly increased after the 7-d immersion (Fig. 9a–d). The fitted electrochemical parameters are listed in Tables 1, 2, and 4. Theoretically, the polarization resistance (Rp) is inversely related to the corrosion rate59. During the 7-d immersion period, the Rp of abiotic Cu and Ni coupons remained around 60 and 25 kΩ cm2, respectively, indicating good corrosion resistance. The corrosion current density (icorr) of abiotic coupons (Fig. 9f) were 1.32 × 107, 8.96 × 108, 1.52 × 106, and 1.08 × 106 A cm−2 corresponding to Cu, Cu with riboflavin, Ni, and Ni with riboflavin, respectively. These results indicated that riboflavin did not affect the electrochemical data of abiotic coupons.

Fig. 9: Electrochemical measurement of the abiotic control.
figure 9

Nyquist and Bode plots of Cu (a, b) and Ni (c, d) coupons, Rp and Rct from fitted EIS parameters (e), Potentiodynamic polarization curves (f) of Cu and Ni after the 7-d immersion in the sterile medium without and with riboflavin. (Error bars stand for the standard deviations from at least three independent coupons).

Table 1 Fitted electrochemical parameters from EIS data of Cu.
Table 2 Fitted electrochemical parameters from EIS data of Ni.
Table 3 Fitted electrochemical parameters from EIS data in Fig. 13.
Table 4 Tafel parameter for Fig. 9f.

Figure 10a shows the open circuit potential (OCP) variation vs. time of Cu and Ni coupons in D. vulgaris media without and with riboflavin during the 7-d incubation period. There was no significant difference in OCP values of Cu electrodes without riboflavin and with riboflavin. The daily OCP values of Ni electrodes with riboflavin were lower than that without riboflavin. The OCP values of Cu electrodes (−750 ± 80 mV, vs. saturated calomel electrode, SCE) were more negative than that of Ni electrodes (−600 ± 50 mV), indicating a stronger corrosion tendency for Cu MIC than for Ni MIC.

Fig. 10: OCP and Rp.
figure 10

Variation of the OCP (a) and Rp (b) of the Cu and Ni coupons during the 7-d incubation in the D. vulgaris media without and with riboflavin. (Error bars stand for the standard deviations from at least three independent coupons).

Figure 10b shows the Rp curves of Cu and Ni coupons in D. vulgaris media without and with riboflavin during the 7-d incubation period. For the trend of Ni Rp values without and with riboflavin, Rp values were closer on the 1st-d (6–8 kΩ cm2) and then reduced over time. The Ni Rp curves with riboflavin were significantly lower than that without riboflavin, indicating that the addition of riboflavin resulted in a more severe Ni MIC. But the Cu Rp curves with and without riboflavin were nearly the same. In addition, the Cu Rp values (0.4–0.7 kΩ cm2) were far lower than that of Ni (2–8 kΩ cm2), corresponding to the much larger weight loss data for Cu coupons in Fig. 6.

Figure 11 shows the Nyquist and Bode plots of Cu and Ni coupons after the 1-d, 3-d, 5-d, and 7-d incubation in D. vulgaris media without and with riboflavin. The Nyquist plots of Cu and Ni electrodes exhibit distinctly different characteristics. The Nyquist plots of Cu without and with riboflavin were almost similar, indicating that riboflavin did not affect the Cu MIC. Unlike the Cu MIC, the radius of the semicircle for Ni electrodes with riboflavin was smaller than that of Ni electrodes without riboflavin, indicating that Ni MIC was accelerated by riboflavin. In the Bode diagrams, the low-frequency impedances of Ni electrodes with riboflavin were lower than that without riboflavin, corresponding to the Rp (Fig. 10b) and weight loss data (Fig. 6). The Bode diagram also shows the constant phase element (CPE) behavior in the low and mid-frequency regions. The time constants in low-frequency and mid-frequency regions correspond to the charge transfer reaction and corrosion product film, respectively59.

Fig. 11: EIS.
figure 11

Nyquist and Bode plots of Cu and Ni coupons during the 7-d incubation in the D. vulgaris media without and with riboflavin: 1-d (a, b), 3-d (c, d), 5-d (e, f), and 7-d (g, h).

The equivalent circuit models are illustrated in Fig. 12a to fit the electrochemical impedance spectroscopy (EIS) data, equivalent circuits I and II for Cu, and equivalent circuit III for Ni. Model I: Rs(QfRf)(QdlRct) is a series circuit that represents the electron transport between the media solution/film and film/Cu interfaces (for 1-d). Model II: Rs(Qf(Rf(QdlRctW))) is a parallel circuit, suggesting the corrosion product deposit on the Cu surface is a poor barrier (for 3-d, 5-d, and 7-d)60. Model III: Rs(Qf(Rf(QdlRct))) is used to describe the response of the media solution/film and film/Ni interfaces (for 1-d, 3-d, 5-d, and 7-d). In equivalent circuit models, Rs, Rf, and Rct stand for solution, film, and charge transfer resistances, respectively. Qf and Qdl represent the film capacitance, and double-layer capacitance, respectively. W is the Warburg impedance, which represents the resistance to mass transfer. The appearance of W indicates the electrochemical reaction process is controlled by diffusion, which may be due to the formation of a relatively dense Cu2S film that inhibits ions from transporting to Cu surfaces for reaction. The nonideal capacitive behavior used CPE instead of capacitance, defined as:

$$Z_{{{{\mathrm{CPE}}}}}(\omega ) = Y_0^{ – 1}(j\omega )^{ – n}(j^2 = – 1)$$

(6)

where Y0 and n are the parameters of CPE (n = 1, an ideal capacitor; 0.5 < n < 1, a nonideal capacitance; and n = 0 represents a perfect resistor), ω is the angular frequency, and j is the imaginary unit.

Fig. 12: Equivalent circuit diagram of EIS fitting.
figure 12

a Equivalent circuit models for EIS fitting, b Variation of Rf + Rct of Cu and Ni coupons during the 7-d incubation in the D. vulgaris media without and with riboflavin. (Error bars stand for the standard deviations from at least three independent coupons).

The selected fitting circuits fit well with EIS data (Fig. 11), and the fitted values are listed in Tables 1 and 2. Rp is the total of Rf and Rct for the equivalent circuits with two-time constants, i.e., Rp = Rf + Rct, the sum of resistances is also a key parameter for MIC37. Figure 12b shows the variation of Rf + Rct values of Cu and Ni electrodes in D. vulgaris media without or with riboflavin. For Ni MIC, the Rf + Rct curve with riboflavin was much higher than that without riboflavin, indicating that riboflavin accelerated the Ni MIC. The variation of Rf + Rct values vs. time was similar to that of Rp (Fig. 10b).

Figure 13 shows the electrochemical responses of D. vulgaris biofilm of 3-d pre-growth on Cu and Ni coupons to riboflavin. The Ni Rp decreased by 45% (from 7.22 to 3.98 kΩ cm2) within 1 h after adding riboflavin, while the Ni Rp without riboflavin decreased by 6.2% (from 7.22 to 6.83 kΩ cm2). After the 1-h period, the Rp value of Ni with riboflavin was 42% lower than that without riboflavin. The response of D. vulgaris biofilm to riboflavin was quite fast, and the variation of Rf + Rct (Fig. 13f and Table 3) is consistent with Rp values (Fig. 13e). By comparison, riboflavin did not alter Cu Rp values significantly within 1 h, which was almost the same as that without riboflavin (~0.51 kΩ cm2). These results also confirmed that the Cu MIC by D. vulgaris was not EET-MIC6.

Fig. 13: Electrochemical responses of the D. vulgaris biofilm of 3-d pre-growth to riboflavin.
figure 13

Nyquist and Bode plots showing biofilm responses on Cu (a, b) and on Ni (c, d) coupons to injection of riboflavin, and the variations of Rp (e), and Rf + Rct from fitted EIS parameters (f). (Error bars stand for the standard deviations from at least three independent coupons).

Figure 14 shows the potentiodynamic polarization curves of Cu and Ni coupons after the 7-d incubation in D. vulgaris media without and with riboflavin. The corresponding Tafel parameters are listed in Table 5. The icorr of Ni increased from 5.27 × 10−6 to 8.39 × 106 A cm−2 (59% increase) with the addition of riboflavin. It was further confirmed that the addition of riboflavin increased the MIC rate of Ni. In contrast, the potentiodynamic polarization curves of Cu without or with riboflavin were almost the same and the icorr values did not differ much, icorr values of Cu had only a slight decrease from 1.05 × 10−5 to 9.54 × 10−6 A cm−2. This result corresponds to the Rp (Fig. 10b) and EIS data (Fig. 11).

Fig. 14: Potentiodynamic polarization and icorr.
figure 14

a Potentiodynamic polarization curves, b icorr analysis of the Cu and Ni coupons after the 7-d incubation in the D. vulgaris media without and with riboflavin. (Error bars stand for the standard deviations from at least three independent coupons).

Table 5 Tafel parameter for Fig. 14.

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