# Optimization of Microalgae-Bacteria Consortium in the Treatment of Paper Pulp Wastewater
## 2 Materials and Methods
### Analytical Methods  
On days 0, 2, 5, 7, 9, 12, and 14, a water sample was collected to determine pH, dissolved oxygen (DO), temperature, chemical oxygen demand (COD), VSS, total nitrogen (TN), ammoniacal nitrogen (NH\({}_{4}\)+-N), nitrite nitrogen (NO\({}_{2}\)\({}^{-}\)N), nitrate nitrogen (NO\({}_{3}\)\({}^{-}\)N), and phosphate phosphorus (PO\({}_{4}\)\({}^{3}\)\({}^{-}\)-P). The temperature, pH, and DO were analyzed using the probes Sentix41 and Oxical and a Multi 3401 meter (WTW, Germany). To assess the concentration in the liquid phase, the water samples were filtered through 0.45 \(\upmu\)m membranes to remove solids. The colorimetric method was used to analyze COD, NH\({}_{4}\)+-N, NO\({}_{2}\)\({}^{-}\)N, NO\({}_{3}\)\({}^{-}\)N, and PO\({}_{4}\)\({}^{3}\)-P, using the RFID model DR3900 technology (Hach, Loveland, CO, USA) and the cuvette tests LCK 114, LCK 303, LCK 342, LCK 339, and LCK 350, respectively. TN and VSS were carried out through standard methods [28]. Chlorophyll (CHL) analyses were also performed in all water samples to monitor the development of microalgae; regardless of the species, this parameter indicates the productivity of algal biomass of the symbiotic process. The analysis of chlorophyll \"a\" and \"b\" was performed according to the methodology indicated by [PERSON] et al. [29] and [PERSON] et al. [30], where an aliquot of 30 mL of mixed liquor from each photobioreactor was centrifuged for 10 min at 4000 rpm. The supernatant was then discarded, and 30 mL of acetone and methanol (2:1 \(v/v\)) were added. The solution was then introduced into a Sonics Materials, Vibracell (Newtown, CT, USA) sonicator for 100 s, resulting in cell breakage and pigment extraction. After sonication, the samples were conditioned in the dark, with aluminum foil around the falcon tubes, for 60 min in a refrigerator at 4 \({}^{\circ}\)C. Sequentially, the samples were centrifuged for 10 min at 4000 rpm at room temperature. Finally, the chlorophyll-a and the chlorophyll-b optical density (OD) was measured at wavelengths of 665 nm, 645 nm, and 630 nm in the supernatant, using a Spectronic Helios Gamma UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Equations (1) and (2) were used for the calculation of chlorophyll \"a\" and \"b\" (mg/L), respectively [29]. Chlorophyll - a = \[\frac{\left[(11.85\times(\text{OD}665)-1.54\times(\text{OD}645)-0.08 \times(\text{OD}630)\right]\times\text{V1}}{\text{V}}\] (1) Chlorophyll - b = \[\frac{\left[(-5.43\times(\text{OD}665)+21.03\times(\text{OD}645)-2.66 \times(\text{OD}630)\right]\times\text{V1}}{\text{V}}\] (2) where chlorophyll-a is the concentration of chlorophyll a (mg/L), chlorophyll-b is the concentration of chlorophyll b (mg/L), OD665 is the wavelength at 665 nm, OD645 is the wavelength at 645 nm, OD630 is the wavelength at 630 nm, V1 is the volume of solvent (2:1) (L) and V is the volume of sample (L). A sedimentation test was also performed for all water samples to assess the biomass's sedimentation capability, following the methodology described by [PERSON] et al. [26]. A 50 mL aliquot of the mixed liquor sample was withdrawn, and its initial optical density (OD) was measured at a wavelength of 650 nm, after the sample was allowed to rest for 20 min before being tested for OD at the same wavelength (the new aliquot was extracted up to a maximum of 2.5 cm from the surface). Equation (3) shows the biomass flocculation efficiency (%):  
\[\text{FE}=\left(1-\frac{\text{F}}{\text{I}}\right)\times 100 \tag{3}\]  
where FE is the flocculation efficiency (%), F is the OD\({}_{650}\) after 20 min of sedimentation and I is the corresponding OD of the homogenized sample's OD\({}_{650}\) (T = 0). Regarding the composition of the biomass, the extracellular polymeric substances (EPS) were extracted according to [PERSON] and Buitron [31]. Initially, 30 mL of mixed liquor was centrifuged at 4000 rpm for 20 min. The liquor was vortexed after the supernatant was removed and 15 mL of saline solution (NaCl) was added.