**5. Methodology**

### **5.1 Materials**

*Renewable and Sustainable Composites*

NPs can produce ROS, and even if they could, many do not produce them all. For example, calcium oxide (CaO) and magnesium oxide (MgO) NPs can only produce O2<sup>−</sup> [34]. ZnO can generate H2O2 and OH<sup>−</sup> but not O2<sup>−</sup> [34], while TiO2 and copper oxide (CuO) NPs can produce all four types of ROS [34]. For the sake of simplicity, the present work focuses only on TiO2 irradiated with UV light and is used as a ROS

Such ROS production induced by the irradiation of TiO2 with UV light is shown in the following equations. Eq. (1) describes the energy absorption and the photocatalytic reaction. Eqs. (2)–(4) depict the photocatalytic redox pathways involved in the generation of an O2<sup>−</sup> and an OH<sup>−</sup> at the reaction between the holes with H2O and the electrons with O2<sup>−</sup> [35]. Eqs. (5), (6) describe the generation of H2O2 by

<sup>+</sup> → *OH*<sup>−</sup>

+ *hVB*

The ROS formation is an oxidative stress mechanism due to the generation of an imbalance between the production of free radicals and the ability of the cell to counteract. Different bacteria have particular ways to protect themselves to some

<sup>−</sup> + 2*H*<sup>+</sup> + *eCB*

<sup>−</sup> + *hVB*

<sup>+</sup> (1)

+ *H*<sup>+</sup> (3)

<sup>−</sup> → *O*2− (2)

<sup>+</sup> → *OH*<sup>−</sup> (4)

<sup>−</sup> → *H*2*O*<sup>2</sup> (5)

<sup>+</sup> + 2*H*2*O* → *H*2*O*<sup>2</sup> + 2*H*<sup>+</sup> (6)

reductive and oxidative pathways, respectively.

*Schematic illustration for the photocatalytic reaction of TiO2.*

*O*<sup>2</sup> + *eCB*

*H*2*O* + *hVB*

*OH*<sup>−</sup>

*O*<sup>2</sup>

2*hVB*

**4.1 Oxidative stress**

*TiO*<sup>2</sup> + *energy* → *eCB*

**78**

source.

**Figure 3.**

For the production of the biocomposite, the following materials were used: chitosan powder (coarse ground flakes, deacetylated chitin, poly(D-glucosamine) CAS number: 9012-76-4), acetic acid (glacial ACS reagent (2.5 L, RABA0010–2.5D1)), and TiO2 (anatase polymorph) purchased from Sigma Aldrich® now Millipore Sigma® (22 nm, nanocrystalline colloidal paste for transparent films, >95% anatase by x-ray diffraction, 60–65% porosity, specific surface area 65–75 m<sup>2</sup> , pH <1, #798495).

To test the antibacterial properties of this renewable biocomposite, *E. coli* (ACTC 25922) and *S. aureus* (ATCC25923), provided by the Biology Department at the University of Puerto Rico—Mayagüez (UPRM), were used. The bacterial medium used to grow them was the Miller's Luria Broth (LB, CAS number: 91079–40-2, tryptone 10 g/L, yeasts 5 g/L, and sodium chloride 10 g/L), provided by Research Product International (RPI). An in-house Millipore filter provided the necessary deionized water. To provide aseptic conditions and for disinfection, common Lysol® diluted in distilled water was used. The use of protective gear, i.e., face masks, laboratory coats, gloves, and safety glasses, was mandatory throughout the entire experimental work. Laboratories in the UPRM Biology Building and Stéfani Building hosted the present research.
