*3.2. Antibiofilm Activity on the TiO2 and Ag-TiO2 Nanocomposite Coatings*

Antibacterial epoxy coatings for antibiofilm properties were tested against *S. aureus* and *E. coli*  under static conditions in glass petri dish with UV-A irradiation, on the surfaces of TiO2 and Ag-TiO2 composites (both with 1 wt% loading). Both *S*. *aureus* and *E. coli* were able to form biofilm on neat epoxy resin surface (negative control) and composites, *i.e*., biofilm formation was independent of the underlying composite substrates. In the absence of TiO2, epoxy resin showed higher growth of biofilm than that of epoxy/TiO2 composite. Anti-boifilm activity appeared to increase significantly for Ag-TiO2 composite.

The biofilm inhibition by composites does not seem to be restricted to specific strains or growth conditions; *E. coli* and *S. aureus* varied in their ability to produce biofilm on the surface of the composites as shown in Figure 6. In all assays, the amount of crystal violet eluted from *E. coli*  biofilms was lower than that of *S. aureus* biofilms, because *E. coli*, being a Gram negative organism binds lesser dye than Gram positive organisms like *S. aureus*. The OD600 of CV eluates from both biofilms was in the range of 0.121 to 2.8. Among the bacterial pathogens, *E. coli* was more susceptible for biofilm inhibition than *S. aureus* on these surfaces.

**Figure 6.** Spectrophotometric analysis (OD600) of solubilized crystal violet of *E. coli* and *S. aureus* biofilm at 18 h irradiation time on the surfaces of TiO2 and Ag-TiO2 composite with similar loading (1 wt%).

To confirm the activity of TiO2/Ag-TiO2 on the surface of nanocomposite for the photokilling, we conducted the experiments under both dark and irradiated conditions as shown in Figure 7, and we found that higher inhibition of biofilm under irradiated conditions as shown in Figure 7b. The Ag-TiO2 composite (1 wt%) showed 24% and TiO2 composite (1 wt%) showed 6% biofilm inhibition of *E. coli* after 18 h of incubation in the dark as shown in Figure 7a. For the same conditions with UV irradiation *E. coli* biofilm showed 56% inhibition for epoxy/TiO2 and 77% inhibition for epoxy/Ag-TiO2, while that of *S. aureus* biofilm showed 43% and 67% ihibition, for epoxy/TiO2 and epoxy/Ag-TiO2 composites respectively. It is, therefore, the bactericidal activity of silver on biofilm that is rendered more likely in the absence of photokilling by Ag-TiO2 with the dark experiment data. However, enhanced antibiofilm response of Ag-TiO2 composite under UV irradiation can be attributed to the silver surface plasmon band favoring UV light absorption along with nanometer sized silver particles which exhibited a striking degree of synergy. The antibacterial feature was diminished for epoxy/TiO2 composite in the dark experiment. However, the bare TiO2 particles which are non-photo-activated on the surface also supported minor antibacterial activity, even in the dark. This is due to direct attack of cells upon contact with TiO2 nanoparticles which disrupt the integrity of the bacterial membrane [31,32]. This is also in agreement with reported experimental findings by Gogniat *et al.* [33] who also showed a loss of bacterial culturability after contact with TiO2 nanoparticles even in the dark. These data show that the nature of epoxy resin makes it suitable host for dispersion of photocatalyst like TiO2 for bacteriacidal activity.

**Figure 7.** Mean values of quadruplicate experiments showing percent inhibition of *E. coli* and *S. aureus* bio-film formation on epoxy/TiO2 and epoxy/Ag-TiO2 composite surfaces calculated relative to the neat epoxy (negative control), under (**a**) dark and (**b**) UV irradiated conditions.

The release of the antimicrobial species (Ag+ , Ag<sup>0</sup> and ROS) from a composite occurs due to the interaction of the diffused water molecules with TiO2 and dispersed silver within the matrix during UV exposure; upon submerging it in the culture media [34,35]. Silver ions resident within the metal oxide nanofiller can diffuse to the surface of the epoxy matrix. The leaching of Ag+ ions was confirmed by AAS analysis of the bacterial media from blank experiments (without inoculums as explained in the experimental section). The Ag+ ion concentration of the same media was determined by atomic absorption spectrophotometer (AAS), which strongly suggests Ag+/Ag0 are associated noncovalently with cross-linked polymeric host and has leached to aqueous medium. By AAS analysis, the silver concentration (Ag+/Ag0 ) in the exposed media for the different epoxy/Ag-TiO2 composite, showed a nonlinear increase that approached a maximum for the composite with 2.0 wt% of Ag-TiO2 loading Table 1.

The valence band "electrons" can be excited to the conduction band (e<sup>í</sup> cb), leaving positive "holes" in the valence band (h+ vb) to form an e<sup>í</sup>/h+ couple that react with aqueous environment and oxygen, to generate reactive oxygen speces (ROS) such as OH**.<sup>í</sup>**, HO2 **.<sup>í</sup>** and O2 **.<sup>í</sup>**, which are responsible for the mechanistic photo-biocidal activity [36,37].The photoexcitation of non-leachably associated TiO2 occurs when it absorbs light equal to or greater than band-gap energy near-ultraviolet light region. While Ag NP and Ag<sup>+</sup> could act as efficient electron scavengers, and significantly enhanced the visible light responsiveness of TiO2 to generate more oxygen free radicals by improving the quantum efficiency of a charge pair generated [35]. At the same time, these oxygen species can reduce Ag+ ions to form Ag nanoparticles. The smaller Ag+ ions can easily penetrate the cell wall and thus can hasten antimicrobial activity.

The attack of Ag+ on disulfide or sulfhydryl (thiol) groups present in the membrane protein result in formation of stable S–Ag bond with –SH groups thus inhibiting enzyme-catalyzed reactions and the electron transport chain that are necessary for biofilm formation [38]. We speculate that the outer membrane of the bacterial cell is attacked by photocatalytic oxidation enabling the antimicrobial metal ions/particles to diffuse to interior of the cell thus becoming much more lethal to the bacterium. Thus, capability of photoactiveTiO2 and leachable silver in destabilizing the biofilm matrix is enhanced by synergistic approach.
