## 2 Materials and Methods
### Flask Culture Experiments  
Initially, the tolerance of duckweed and microalgae to RW was studied. Treatment cultures consisted of mixing RW with distilled water (DW) in different proportions, specifically 10%, 20%, 30%, 40%, and 50% (\(\
icefrac{\
u}{\
u}\)) (duckweed and microalgae did not survive above 50% and 30%, respectively). The control culture medium consisted of BG11 medium for microalgae, while HSH medium was utilized for _Lenna minor_. Treatment cultures with RW were not supplemented with nutrients from BG11 and HSH. The cultivation conditions matched those described in Section 2.1. In the case of duckweed, 400 mL circular glass containers with a working volume of 200 mL were utilized. The factor RW to DW ratio was investigated for three initial population surface densities of _Lenna minor_: 2.50, 3.75, and 5.00 g dm\({}^{-2}\). The effect on growth was assessed based on the final wet biomass yield. Briefly, at the end of each culture experiment, all duckweed plants from each treatment were harvested. The harvested plants were washed with distilled water to remove surface impurities and then subjected to forced ventilation with cold air to remove excess surface water. Subsequently, the biological material was weighed using a high-precision analytical balance (A&D GH-200, Tokyo, Japan). The biomass yield measurements were expressed in terms of wet or fresh weight (FW). The total chlorophyll content (\(Chl_{total}\)), which comprises chlorophylls a and b, was determined following a modified protocol based on [PERSON] et al. (2019). Briefly, wet duckweed fronds were prepared as described above for the whole plants. Subsequently, 1 g wet weight of this biological material was weighed using a high-precision analytical balance, followed by crushing in a mortar with 10 mL of 99.9% absolute anhydrous ethanol (denatured SDA-40B, Merck, Darmstadt, Germany). The resulting extract was filtered through a 0.45 \(\upmu\)m PTFE-L membrane filter (Cobetter, Nantong, China) and transferred to a 15 mL Falcon(tm) tube, where it was left to stand for 48 h at 4 \({}^{\circ}\)C in the absence of light. The suspension was then centrifuged at 23,478 RCF-max for 10 min at 4 \({}^{\circ}\)C (Rotanta 460R, Hettich Zentrifugen, Tuttlingen, Germany). Subsequently, the absorbance of the supernatant was measured using a UV-visible spectrophotometer (UH-5300, Hitachi, Japan) at the wavelengths of 649 and 664 nm, as recommended by [PERSON] (2010), with a 99.9% ethanol solution used as the blank. Chlorophyll concentrations were calculated following the equations proposed by [PERSON] (2010) (see Section 2.6), and the results were expressed in mg of \(Chl_{total}\) per gram of wet weight of from tissue (mg g\({}^{-1}\) FW). The assays were conducted using duckweed plants acclimated to each culture medium (for at least one month with several passages of fresh medium) to ensure the adaptability of the cultivated species to the RW-based culture medium. For the microalgae, the cultures were carried out in 300 mL Erlenmeyer glass flasks with a 250 mL working volume, as described elsewhere (13), following the same environmental conditions as described in Section 2.1. The influence of different culture medium formulations on growth and biomass yield was studied. The assays were carried out using cells acclimated to each culture medium (at least two months with several medium passages) to ensure the long-term adaptability of the cultivated species to the wastewater-based culture medium. The media were inoculated using exponentially growing cells. The experiments started at a mean initial cell concentration of 1.0 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\). The initial pH for all cultures (_Lenna minor_ and microalgae) was adjusted to 7.2 using a 2 M NaOH solution. The pH was allowed to evolve freely in all cultures. The experiments were carried out in triplicate.