*3.3. CLSM*

To evaluate the inhibitory effect of BDMDAC to biofilm formation, CLSM was used for observation. Figure 4 shows the CLSM images of the 304SS coupons after 14 and 28 days of immersion in various solutions. In the culture medium without *D. desulfuricans* inoculation and BDMDAC, no growth was observed (Figure 4a,d). After incubation with *D. desulfuricans*, numerous green dots were observed, as shown in Figure 4b,e. The cells in the biofilm were stained green after staining, suggesting that most of the cells were alive. Furthermore, Figure 4b,e show that the thickness of biofilm increased to 18 and 40 μm for 14 and 28 days of immersion, respectively. In the prescence of BDMDAC, the thickness of biofilm was less than 20 μm, even after 4 weeks. Furthermore, most cells in the biofilm were now dead, as indicated by the red stains (Figure 4c,f). Notably, the biofilm grown on the 304SS coupons in the BDMDAC solution was not visible in the previous SEM images (Figure 3c,f). This can be attributed to the fact that killed bacteria or disrupted biofilm due to BDMDAC addition are easily removed during the cleaning process of SEM sample preparation.

**Figure 4.** CLSM 3D images of the 304SS coupons after different immersion times in various solutions: (**a**) blank for 14 days; (**b**) SRB for 14 days; (**c**) BDMDAC for 14 days; (**d**) blank for 28 days; (**e**) SRB for 28 days; and (**f**) BDMDAC for 28 days.

#### *3.4. Potentiodynamic Polarization*

Figure 5 illustrates the potentiodynamic polarization curves of coupons under different immersion environments after 14- and 28-day immersion periods. The values of *Ecorr* and *Icorr* according to the potentiodynamic polarization curve are listed in Table 2. Table 2 shows that the corrosion current densities *Icorr* in the presence of bacteria were higher than that in the control medium for each corresponding exposure time, which suggests the occurrence of MIC. The *Icorr* value was highest in the presence of *D. desulfuricans*. Furthermore, the current density of the anodic branch in the 304SS coupons in the SRB solution was higher than that of those immersed in blank solutions for each corresponding immersion time. These observations can be attributed to the aggressive role of bacteria in enhancing the corrosion of stainless steel by destroying the oxide layer on stainless steel [54]. The *Icorr* value and current density of the anodic branch for the 304SS coupons immersed in the BDMDAC solution significantly decreased compared with coupons after 14 and 28 days of immersion in the SRB solution. These results indicate the ability of BDMDAC to protect stainless steel substrate against *D. desulfuricans*. Notably, the potentiodynamic polarization curves of coupons exposed to the BDMDAC solution still exhibited a little positive shift compared with those of the blank solution curves. This observation can be attributed to the fact that the bacterial adhesion and biofilm formation at very beginning of immersion test could affect the physical and chemical conditions of 304SS coupons, and result in adverse effects on their corrosion performance. Figure 6 shows the OM images of 14-day immersed coupons after potentiodynamic polarization curve measurements. As can be seen, the obvious localized corrosion was found on the 304SS coupon in the SRB solution (as indicated by red arrows in the Figure 6b). This can be attributed to the MIC effect, leading to the deterioration of passive film by sulfide produced by SRB [42] after being immersed in the SRB solution. These results provide considerable support for our potentiodynamic polarization curve results that the lower current density of the anodic branch and poor passivity were found on the 304SS coupon immersed in the SRB solution.

**Figure 5.** Potentiodynamic polarization curves for the 304SS coupons after 14 days (**a**) and 28 days (**b**) of immersion in various solutions.


**Table 2.** Corrosion potential and corrosion current density measured according to potentiodynamic polarization curves after 14 and 28 days of immersion.

**Figure 6.** OM images of 304SS coupons after potentiodynamic polarization curve measurements. These surfaces were initially exposed to (**a**) blank for 14 days; (**b**) SRB for 14 days; and (**c**) BDMDAC for 14 days.

#### *3.5. Limitations of This Study*

Electrochemical techniques include potentiodynamic polarization are useful for examining the corrosion performance of metallic substrates. However, some limitations of electrochemical techniques for investigating microbial corrosion have been proposed. Javaherdashti has mentioned that large polarizations could be deleterious to micro-organisms in the biofilm [55]. However, it should be noticed that the two examples provided in this reference focused on cathodic protection and its effect(s) on MIC. A possible mechanism is that the application of cathodic potential will increase local pH (due to ORR reaction: O2 + 2H2O + 4e− → 4OH−) and result in inhibiting the bacterial reproduction of microbes in such a high alkaline environment [56,57]. In addition, the time staying in the cathodic polarization potential is another important issue to study the effects of cathodic polarization on MIC. Little et al. [58] and Romero et al. [59] had polarized their samples at –400 mV (vs. SCE) for 72 h and –1000 mV (vs. SCE) for 3–72 h, respectively, to investigate the cathodic polarization effects on MIC. In contrast, the length of sample staying in the cathodic polarization potential is only 500 s (from –500 mV to 0 mV vs. open circuit potential (OCP), scan rate: 1 mV/s) in the present study. As a result, it is believed that the electrochemical experimental setup used in this study is likely to have less influence on the microbial environment.

On the other hand, potentiodynamic polarization curves were not periodically made during the immersion test in the present study. These measurements were applied and started applying potential on the 304SS coupons only after 14 or 28 days of immersion. Although the bacterial activity may be influenced during the polarization test, MIC-induced changes in the electrochemical conditions of 304SS coupons during 14 or 28 days of immersion had resulted in adverse effects on their corrosion performance. The potentiodynamic polarization curve measurement of coupons under different corrosion conditions (with and without bacteria) after different exposure times had also been used in numerous relevant studies [5,42–44]. In addition, the coupons for 14- and 28-day immersion tests were prepared in the different bottles, indicating the polarization potential applied on the 14-day immersed coupons would not have influence on the bacteria and culture medium in 28-day immersed ones. For all the above reasons, the electrochemical analysis used in this study gives useful information for research purposes.
