## 4 Discussion  
Many bacteria are known to form biofilms on material surfaces; this phenomenon is responsible for many serious problems. Thus, the elimination of bacteria from material surfaces is of great interest for both industry and daily life [1; 2; 3; 4; 5]. Therefore, the objectives of this study were to identify factors that influence, and find a facile way to reduce, bacterial adhesion on SS. Nafion, which is commonly  
Figure 6: SEM images of **(a)**_B. subtilis_ biofilms on half of SS disc without coating, and **(b)**_B. subtilis_ biofilms on the half of SS disc with 1% Nafion coating.  
Figure 7: Antibacterial ability of a reused Nafion-coated SS surface: **(a)** biofilm on a half Nafion-coated SS disc for the first antibacterial test, and **(b)** biofilm on the reused half of the Nafion-coated SS disc.  
used as a proton exchange membrane, was chosen because of the biocompatibility and its negatively charged polymeric blocks [69; 72; 73]. It has been proven that Nafion is able to hinder the formation of biofilms by _E. coli_, i.e., a typical G\({}^{-}\) bacteria [70]; however, the antibacterial ability of Nafion against G\({}^{+}\) bacteria has not yet been reported.  
For this work, it was hypothesized that Nafion would reduce the adhesion of G\({}^{+}\) bacteria. Herein, SS surfaces were coated with 1% Nafion, and the biofilms of the G\({}^{+}\) bacteria _B. subtilis_ that formed on both coated and uncoated SS surfaces were evaluated. According to the results of this study, the Nafion-coated surface reduced the adhesion of _B. subtilis_ by 75% compared with the uncoated surface. As reported by several studies, the antibacterial efficacies of materials differ with G\({}^{+}\) and G\({}^{-}\) bacteria [56; 61; 62]. However, comparing the results of our previous study [70], Nafion-coated surfaces have been shown to have similar inhibitory efficacy on the formation of biofilms by _E. coli_ and _B. subtilis_, which indicates that the differences in the bacterial cell envelopes of G\({}^{+}\) and G\({}^{-}\) bacteria do not significantly influence the antibacterial ability of Nafion-coated SS surfaces.  
In order to identify factors that influence the adhesion of _B. subtilis_ to Nafion-coated SS surfaces, the toxicity of Nafion against _B. subtilis_ and the surface properties of SS were investigated. The biocompatibility of Nafion has been verified by many studies [72; 73]. As shown by the results of this study, Nafion had no significant toxicity on _B. subtilis_. Therefore, we deduced that Nafion-coated SS surfaces inhibit biofilm formation by resisting bacterial attachment rather than killing the bacteria.  
Furthermore, previous studies have indicated that antibacterial properties are related to surface characteristics [17; 18]. This work focused on the effects of surface roughness, wettability and electrostatic interactions. The roughness of SS discs before and after coating with 1% Nafion showed that the average Ra for both was about 0.27 \(\upmu\)m, which means that the surface roughness of the SS discs before and after coating was comparable. The hydrophilicity of SS surfaces was estimated by contact angles (Figure 3); after coating with 1% Nafion, the SS surfaces became more hydrophobic. As agreed by most researchers [19; 28; 29; 30], bacteria prefer to adhere to hydrophobic surfaces, rather than hydrophilic surfaces; thus, Nafion-coated SS surfaces will attract more bacteria than uncoated surfaces because of their increased hydrophobicity. However, as mentioned, Nafion-coated SS surfaces showed dramatically reduced _B. subtilis_ adhesion in the present study. As bacteria are almost always negatively charged in aqueous suspension, and Nafion-coated SS discs surface are negatively charged when exposed to solutions because of the sulfonic acid groups in Nafion [69; 71], we propose that Nafion-coated surfaces resisted bacterial adhesion by electrostatic repulsion. These results suggest the importance of electrostatic interactions in overcoming the adhesion of _B. subtilis_ to Nafion-coated SS.