## 2 Materials and Methods
### Evaluation of the Bioactive Potential
#### 2.3.2 Determination of the Bioactivity of Microalgae Extracts  
Solutions were prepared with the extracts from Section 2.3.1 at 20 mg/mL. * Total Phenolic Content (TPC) The total phenolic content (TPC) was determined using the Folin-Ciocalteu method, following the procedure described by [PERSON] et al. [34]. Absorbance was measured at 765 nm using a microplate reader (Synergy H1, Biotek, Winooski, VT, USA) in a 96-well microplate ([PERSON], Numbrecht, Germany). Gallic acid was used as the standard for the calibration curve, and results were expressed as milligrams of gallic acid equivalents per 100 mg of extract dry weight (mg GAE/100 mg DW). Three independent analyses were performed for each of the triplicates. * Antioxidant Activity (AA) The AA of the extract solutions mentioned above (20 mg/mL) was determined using three different assays. 1. ABTS The ABTS (2,2\({}^{\prime}\)-azinobis(3-ethylbenzothazoline-6-sulphonic acid)) assay was performed as described by [PERSON] et al. [34]. Trolox was used as the standard for the calibration curve, and the results were expressed as micromoles of Trolox equivalents per 100 mgof extract dry weight (\(\mu\)mol TE/100 mg DW). Three independent analyses were performed for each of the triplicates. 2. DPPH  
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was carried out following the procedure described by [PERSON] et al. [34]. Trolox was used as the standard for the calibration curve, and the results were expressed as micromoles of Trolox equivalents per 100 mg of extract dry weight (\(\mu\)mol TE/100 mg DW). Three independent analyses were performed for each of the triplicates. 3. ORAC  
The ORAC assay was performed as described by [PERSON] et al. [34]. The results were expressed as \(\mu\)mol of Trolox equivalent per one hundred milligrams of extract dry weight (\(\mu\)mol TE/100 mg DW). Three independent analyses were performed in each of the triplicates. * Antimicrobial activity  
With some modifications, the protocol of [PERSON] et al. [35] was used to assess the antibacterial activity of the microalgal extracts. In summary, the procedure comprises five principal steps. The first step involves preparing a bacterial suspension and seeding isolated bacteria onto Mueller-Hinton agar nutrient medium (Biokar Diagnostics, Allonne, France). The Petri dishes are then incubated in an oven at 37 \({}^{\circ}\)C for 18-24 h. Three to five morphologically similar colonies are selected and suspended in the same liquid medium (Mueller-Hinton broth; Biokar Diagnostics, Allonne, France), with vortex agitation. The bacterial suspension is adjusted to 10\({}^{8}\) CFU by adding bacterial colonies or liquid medium as necessary, aiming for a solution absorbance between 0.08 and 0.13 (excitation wavelength 625 nm, Shimadzu UV mini 1240, Tokyo, Japan). In the second step, the microalgal extract solutions and serial dilutions are prepared in a laminar flow cabinet (Telstar Bio-II-A, Terrassa, Spain) by dissolving the extracts in liquid medium. A 96-well microplate is used for the microdilution assay. Each well receives 50 \(\mu\)L of microalgal extract solution and 50 \(\mu\)L of bacterial suspension. Two controls are included: a positive control containing 50 \(\mu\)L of bacterial suspension and 50 \(\mu\)L of liquid medium, and a negative control with 100 \(\mu\)L of liquid medium alone. Before addition, the bacterial suspension (10\({}^{8}\) CFU) is diluted 1:100 in the liquid medium. The microplate is incubated at 37 \({}^{\circ}\)C for 16-20 h.  
The third step is the confirmation of CFU present in the bacterial suspension. Before incubation of the microplate, 10 \(\mu\)L from the positive control is transferred to an Eppendorf tube, mixed with 990 \(\mu\)L of Mueller-Hinton broth, and serially diluted 1:10. Then, 100 \(\mu\)L of the final dilution is seeded onto a Petri dish and incubated at 37 \({}^{\circ}\)C for 16-20 h. The number of colonies is counted, and the assay is deemed accurate if approximately 50 colonies are present. The fourth step is the reading of results.