## 3 Results and Discussion  
### Metabolic Response and Connections with Isotopic, Spectrophotometric, and Monitoring Data  
With the experimental setup investigated in this study, it was possible to compare the biomass growth response across the five environmental conditions shown in Table 2. Figure 3 shows, for W and A, respectively, the biomass growth trends in terms of surface density (expressed as grams of dry algal biofilm per unit area of cultivation) in the five tests. Upon initial observation, the operational conditions set in Exp3 proved to be the most effective for both W and A, resulting in significantly higher growth at the end of the 15-day observation period. Specifically, with W, a final average surface density of biomass of 25.4 g/m\({}^{2}\) was recorded, which is a +91% increase compared to the average value of 13.3 g/m\({}^{2}\) obtained from the other four experiments (Figure 3). With the A culture medium, Exp3 recorded a surface density 56% higher than the average obtained in the other four tests (19.6 vs. 12.6 g/m\({}^{2}\)). With regard to the experimental conditions that were explored in the five experiments, it appears that temperature variations within the range investigated in Exp1 and Exp2 did not result in substantial differences in biomass growth (Figure 3). It seems that the transition from Exp2 conditions to those of Exp3, with the introduction of a higher intensity light source, proved to be a successful strategy, leading to the previously mentioned final yields. In the subsequent two tests (Exp4 and Exp5), no substantial differences were observed between the semi-batch mode of Exp4 and the continuous mode of Exp5. However, it seems that the choice to provide fresh medium with progressively higher frequency than  
Figure 3: Time trend of biomass grown with culture medium A (Allen medium) and W ( wastewater), as surface density. the batch mode of Exp3 was counterproductive, leading to less satisfactory final yields (Figure 3). One possible explanation for this result is that, within this cultivation system and given the nutrient bioavailability in the growth media, the algal community might have adopted a metabolic strategy that meets its needs without requiring immediate access to fresh nutrients. Specifically, the microalgal community in this system might require an initial period to adapt to the growing conditions, during which it might establish a dynamic chemical equilibrium with the culture medium, and then progressively exploit all available resources [31]. [PERSON] et al. [32] indicated that microalgae require a certain acclimation period when exposed to environments with higher CO\({}_{2}\) concentrations than those to which they are used. In this context, the continuous addition of fresh culture medium--potentially containing increased levels of dissolved CO\({}_{2}\)--may have triggered a transient adaptation phase, temporarily influencing cellular metabolism and growth dynamics. This phenomenon could explain the observed interference, as sudden fluctuations in CO\({}_{2}\) availability may affect photosynthetic efficiency, carbon assimilation rates, and intracellular pH homeostasis, all of which play a crucial role in microalgal adaptation to environmental changes. Compelling evidence supporting this interpretation emerged from EA-IRMS and spectrophotometric investigations. The first revealed the presence of interaction mechanisms with matrices external to the biomass; on the other hand, the second seemed to suggest a temporary equilibrium in the concentration of ionic species in solution. Analysing the results of the two methodologies in detail and starting with the EA-IRMS analysis, Table 3 provides a summary of the results obtained, including the elemental composition of carbon and nitrogen (%) and the isotopic signals of the analysed matrices.