## 4 Discussion  
### Effects of the Environmental Parameters on Microalgae Growth and Metabolism  
Winter conditions can be a challenge for WW phytoremediation. Several studies report that both exposure to simulated winter conditions [37] and outdoor cultivation during the cold season [56; 86; 87] result in lower growth rates and limited nutrient removal in microalgae [50; 51]. Interestingly, in the outdoor setup, no significant biomass production was recorded at the end of the experiments, while in the indoor setup, the concentration of dry biomass at the end of cultivation was slightly but significantly higher than the initial value. These results suggest that other factors, but not temperature, influence microalgae growth. Indeed, in addition to the difference in average temperature in indoor and outdoor conditions, the indoor temperature remains stable over time while it is externally variable in outdoor conditions. Probably the reduced photoperiod (and low light availability in general) could be the main cause of the negative effect on microalgal biomass production in both experimentations [40; 55; 88; 89; 90]. Similar results have been reported by other researchers, highlighting that photoperiod is a crucial parameter to consider for microalgae cultivation during cold seasons [37; 40]. However, it cannot be excluded that some differences in the experimental responses are due to the different types of the two photobioreactors (e.g., the cultivation volumes) [46]. Cold stress can reduce photosynthetic activity either by reducing the irradiance threshold for photoinhibition or by slowing down the repair of the photosynthetic apparatus [52; 55; 91]. In the outdoor experiment, severe photoinhibition occurred, as indicated by the decrease in PSII maximum quantum yield [92]. Interestingly, after 14 days the F\({}_{\text{V}}\)/F\({}_{\text{M}}\) value increased, although it remained below the optimal value of 0.6 [84], suggesting acclimation to the new growth conditions. Adaptation to a low-light environment is also suggested by the decrease in the Chl _a/b_ ratio and the increase in the Chl/Car ratio at the end of the outdoor experiment [93; 94]. It is also important to note that in the indoor experiment, the F\({}_{\text{V}}\)/F\({}_{\text{M}}\) remained stable for the entire experiment, probably due to the absence of such a severe temperature as in outdoor cultivation [52; 55; 95]. Adaptation to the new light environment may also be suggested in the indoor experiment, considering the variations in the Chl/Car ratio [93; 94; 96]. Considering the trends of pigment concentrations in both cultures, it is possible to assume that the initial decrease was due to adaptation to new culture conditions and reduced shading effect, while the concentrations at the end of the experiments were affected considerably by the photoperiod [97]. Different algal species accumulate more pigments \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Parameter** & **Unit** & **Day 0** & **Day 3** & **Day 20** \\ \hline COD & O\({}_{2}\) mg L\({}^{-1}\) & 61 & 62 & 59 \\ BOD\({}_{5}\) & O\({}_{2}\) mg L\({}^{-1}\) & 13 & \textless{}10 & \textless{}10 \\ Total suspended solids & mg L\({}^{-1}\) & 32 & 26 & 27 \\ _Escherichia coli_ & CFU/100 mL & 36,000 & 2600 & 72 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Media composition at the end of the outdoor experiment. For analyses after the inoculum and on the 3 rd day, the media was collected through centrifugation. On the last day, the media was collected before the manual resuspension, and it was not centrifuged. with different combinations of irradiance and photoperiod, with some preferring high irradiance and reduced hours of light, while others prefer a lower irradiance and more hours of light [97; 98; 99]. However, it is also important to consider the negative effect that cold stress may have had on the pigment concentration of the microalgae cultivated outdoors [90].