## 2 Materials and Methods  
### Microalgae Cultivation  
The microalgal strain employed in this study was isolated from secondarily treated municipal WW and described as _Chloorella_-like as reported in [PERSON] et al. [67]. The microalgae were provided by Alga&Zyme Factory s.r.l. (Ferrara, Italy) and were maintained in bag cylindrical photobioreactors (22 \({}^{\circ}\)C, 90 \(\upmu\)molphotons m\({}^{-2}\) s\({}^{-1}\) PAR, 16-8-h light-dark photoperiod) in BG11 medium with N and P content modified to match the ammonium  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Algal Species** & **Cultivation System** & **Time Period and Location** & **Ref.** \\ \hline \multirow{6}{*}{_Scenedesmus obliquus_} & High-rate algal pond and an & \multirow{6}{*}{From October 2011 to March 2012} \\  & Airlift tubular & & \\  & photobioreactor both operated & & in South Spain. \\  & in continuous. & & \\ _Chloella_ sp., & & & \\ _Scenedesmus_ sp. and & & & \\  a pennate diatom & & & \\  Co-culture of _Chloella_ sp., & & & \\ _Stigeconloium_ sp., & & & \\ _Nitzschia_ sp. and _Nauicula_ sp. & & & \\ _Scenedesmus_ sp. and _Chloella_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ _Scenedesmus_ sp. & & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Prototype or large-scale experiments regarding applications of microalgae in ASP during the winter season. Algal species, cultivation systems, and procedures, along with time period and location are listed.  
and the total P concentrations of the WW from which the algae were isolated: 380 mg/L for NH\({}_{4}\)\({}^{+}\)-N and 110 mg/L for PO\({}_{4}\)\({}^{3-}\)-P. The final recipe was NH\({}_{4}\)Cl 1.45 g/L, K\({}_{2}\)HPO\({}_{4}\) 0.62 g/L, MgSO\({}_{4}\)\(\times\) 7H\({}_{2}\)O 0.075 g/L, CaCl\({}_{2}\)\(\times\) 2H\({}_{2}\)O 0.036 g/L, Na\({}_{2}\)CO\({}_{3}\) 0.02 g/L, EDTA-Na\({}_{2}\) 0.001 g/L, citric acid 0.006 g/L, ammonium ferric citrate 0.006 g/L, and BG-11 Trace Metals Solution (H\({}_{3}\)BO\({}_{3}\) 2.86 g/L, MnCl\({}_{2}\)\(\times\) 4H\({}_{2}\)O 1.81 g/L, ZnSO\({}_{4}\)\(\times\) 7H\({}_{2}\)O 0.222 g/L, NaMoO\({}_{4}\)\(\times\) 2H\({}_{2}\)O 0.39 g/L, CuSO\({}_{4}\)\(\times\) 5H\({}_{2}\)O 0.079 g/L, Co(NO\({}_{3}\))\({}_{2}\)\(\times\) 6H\({}_{2}\)O 0.0494 g/L) 1 mL/L. The algae used for the inoculation were at a stationary growth phase with an OD\({}_{750}\) = 1.604. Absorbance at 750 nm was measured with a Pharmacia Ultrospec 2000 UV-Vis spectrophotometer (1-nm bandwidth; Amersham Biosciences, Piscataway, NJ, USA).  
The effluent of the thickening step of sludge treatment was collected in November 2021 from Hera SpA (Holding Energia Risorse Ambiente) WWTP located in Ferrara, Italy (44\({}^{\circ}\)51\({}^{\prime}\)49\({}^{\prime\prime}\) N, 11\({}^{\circ}\)37\({}^{\prime}\)47\({}^{\prime\prime}\) E). The fresh WW was composed of a solid residue precipitating to the bottom of the tank and an opaque supernatant (OD\({}_{750}\) = 0.14). The WW was not mixed; only the supernatant was used as a culture medium for this study. The composition of the WW supernatant, determined using standard certified methods for water quality analyses, is shown in Table 2.  
To test the ability of this microalga to remove nutrients and grow during winter, two different setups were designed (see below). Both experiments took place in December 2021 and lasted 20 days. Samples were collected on days 0, 1, 2, 3, 6, 7, 10, 14, 17, and 20 of both experiments.