## 2 Materials and Methods
### Phytoremediation Analysis  
Three aliquots of culture medium were collected from both cultures through centrifugation (4500\(\times\)\(g\), 10 min) for the quantification of nitrate (N-NO\({}_{3}\)\({}^{-}\)), ammonium (N-NH\({}_{4}\)\({}^{+}\)), and phosphate (P-PO\({}_{4}\)\({}^{3-}\)) using a flow-injection autoanalyzer (Flowsys, Systea SpA, Company, Roma, Italy). The detection limit for the nitrate and phosphate analyses was 0.02 mg L\({}^{-1}\), while for the ammonium was 0.1 mg L\({}^{-1}\). The same method was used to evaluate the percentage of N and P in the microalgal biomass. For this purpose, an appropriate amount of biomass was collected on days 0, 6, 14, and 20 of both experiments through centrifugation (4500\(\times\)\(g\), 10 min) and dried through evaporation. Then, at least 20 mg of dried biomass was extracted from each sample in 3 mL of selenous H\({}_{2}\)SO\({}_{4}\) at 420 \({}^{\circ}\)C and analyzed as previously described. Given the large volumes required, only one replica was performed for each test. The removal efficiency in percentage (RE, %) was calculated according to the following equation:  
\[\text{RE}\left(\%\right)=\left[\text{(C}_{0}-\text{C}_{1})/\text{C}_{0}\right] \times 100 \tag{1}\]  
where C\({}_{0}\) and C\({}_{1}\) are the nutrient concentrations after the algal inoculum (0) and one day after the inoculum, respectively [67]. To estimate the quantity of nitrogen that was removed from the medium and accumulated inside the microalgae biomass, firstly, the nitrogen content in biomass per liter of culture (N\({}_{\text{B}}\), g\({}_{\text{N}}\) L\({}^{-1}\)) was calculated as follows:  
\[\text{N}_{\text{B}}=\text{\%}_{\text{N}}\times\text{DW} \tag{2}\]  
where %N is the percentage of N inside the biomass (g\({}_{\text{N}}\) in 100 g\({}_{\text{DW}}\)); DW is the dry weight (g\({}_{\text{DW}}\) L\({}^{-1}\)). Then, total N concentration in the medium at the beginning of the experiments (N\({}_{\text{T}0}\)) and the percentage of N removed from the medium and accumulated inside the biomass (N\({}_{\text{A}}\)) were calculated as follows:  
\[\text{N}_{\text{T}}=\text{N-NH}_{4}{}^{+}+\text{N-NO}_{3}{}^{-} \tag{3}\]  
\[\text{N}_{\text{A}}\left(\%\right)=\left[\text{(N}_{\text{B}20}-\text{N}_{ \text{B}0})/\text{(N}_{\text{T}0})\right]\times 100 \tag{4}\]  
where N\({}_{\text{B}20}\) and N\({}_{\text{B}0}\) represent the nitrogen content in biomass at the end of the experiment and on the day of inoculum, respectively. Finally, the nitrogen that was removed from the medium and was not accumulated inside the biomass, called volatilized nitrogen (N\({}_{\text{V}}\)), was calculated as follows:  
\[\text{N}_{\text{V}}\left(\%\right)=\left[\text{(N}_{\text{T}0}-\text{N}_{ \text{T}20})/\text{(N}_{\text{T}0})\right]\times 100-\text{N}_{\text{A}} \tag{5}\]  
where N\({}_{\text{T}20}\) and N\({}_{\text{T}0}\) are the sums of N-NH\({}_{4}{}^{+}\) and N-NO\({}_{3}{}^{-}\) concentrations at end and at the beginning of the experiments, respectively. For pH measurement, a Jenway mod. 3510 (Staorshire, Stone, UK) bench pH meter was employed. After the inoculation, on the third and last days of the outdoor experiment and the last day of the indoor experiment, the culture media were analyzed to investigate the concentrations of _E. coli_, BOD\({}_{5}\), COD, and TSS. The high volumes required for these analyses allowed them to be carried out for indoor cultivation only at the end of the experiment. Since the algal cells naturally deposited, as described in Section 3.1, leaving a clear supernatant, culture medium was collected before manual resuspension. Samples for the outdoor culture were collected after the inoculation of algae, on the third day and at the end of cultivation. For analyses of the samples gathered after the inoculation and on the third day, culture media were collected through centrifugation (2500\(\times\)_g_, 10 min) to separate algal biomass from the medium.