## 3 Results and Discussion
### Metabolic Response and Connections with Isotopic, Spectrophotometric, and Monitoring Data
This source could be atmospheric air (for photosynthesis), which typically has \(\delta^{13}\)C values around \(-\)8% [38] and can circulate freely in both growth chambers through the holes in the base and cover of the PBR. This result suggests the predominance of photosynthetic processes in both growth conditions, as evidenced by the average \(\delta^{13}\)C value of approximately \(-\)26% (Table 3) observed throughout the study period, which is  
Figure 4: Time trend of C and N concentration in biomass grown with A (Allen medium) and W (wastewater) sources. typical of terrestrial plants with a C3 metabolism [39]. In line with these findings, ref. [40] confirmed the presence of photosynthetic pathways in microalgal biomass similar to those in C3 terrestrial plants. Regarding nitrogen, the assimilation mechanisms employed by microalgae rely on the transmembrane transport of inorganic nitrogen from the aquatic environment [41]. Consequently, while no significant alterations of isotopic signal (R\({}^{2}\) = 0.3) were observed in the A standard (as expected), the increase in \(\delta^{15}\)N values observed in W (R\({}^{2}\) = 0.8) (Figure 5) correlates with the increase in %N observed in Figure 4, indicating a clear uptake of nitrogen by microalgae from the wastewater source, the latter being characterised by a \(\delta^{15}\)N signal much higher than that of the initial biomass (Table 3). It is also worth noting that the \(\delta^{15}\)N signal observed in the wastewater medium is consistent with the presence of fertilisers and organic nitrogen [42], which accurately reflects the fact that the wastewater treatment plant supplying the effluents used in this study is situated in a rural area with extensive agricultural and livestock activities. The data obtained from spectrophotometric analyses of ammonium, phosphate and nitrate content in the culture media add interesting insights to the results of EA-IRMS analyses. When examining the behaviour of these ionic species in culture medium W, no significant trend was observed over the days of observation (Figure 6). Instead, their concentrations fluctuated around average values of 32.5 \(\pm\) 2.8 mg/L for NH\({}_{4}\)\({}^{+}\), 2.3 \(\pm\) 0.5 mg/L for PO\({}_{4}\)\({}^{3-}\), and 0.6 \(\pm\) 0.1 mg/L for NO\({}_{3}\)\({}^{-}\). Similar fluctuations were also observed in the standard culture medium A (data available in the Supplementary Materials). Figure 5: Time trend of \(\delta^{13}\)C and \(\delta^{15}\)N isotope ratios in biomass grown with A (Allen medium) and W (wastewater) sources. This trend, observed in both A and W, may indicate the presence of complex physico-chemical and biological mechanisms regulating nutrient availability within the culture system. Since in Exp3 the culture medium was continuously recirculated and not replenished throughout the entire observation period, these fluctuations cannot be attributed to external nutrient inputs. Instead, the data suggest an internal dynamic equilibrium where multiple factors contribute to the stabilisation of ionic species concentrations. The pattern observed in this study aligns with existing knowledge on microalgal nutrient metabolism. Various studies have demonstrated that the assimilation and release of nitrogen and phosphorus in microalgae are strongly influenced by environmental parameters such as pH, temperature, light exposure, and the ionic balance within the culture medium [43]. When intracellular concentrations of NH4+ and NO2- surpass the cells' capacity for assimilation, microalgae may expel these ions into the surrounding medium as a detoxification response to avoid nitrogen stress [44]. While ammonium is generally the preferred nitrogen source for microalgal growth, many species including _Galdieria_ spp. can also utilise organic nitrogen compounds, particularly urea, amino acids, and small peptides, which are commonly present in wastewater [43; 45; 46; 47].