## 3 Results and Discussion
### SEM Characterisation and Projections of Biomass Yields Obtainable from the System  
In addition to the metabolic investigations, two important focuses of this study were (i) the potential for reusing biomass in biotechnological applications and (ii) the long-term biomass yields. Regarding the first aspect, as suggested in Section Rationale Behind the Selection of Twin-Layer Cultivation Technology, the application potential increases with the level of purity of the harvested biomass. Here, the TL-PBR used in this study allows the production of homogeneous and uncontaminated biomass within the observation period. SEM analyses of the biomass (Figure 8) showed that the algal biofilm had a very homogeneous structure, free from foreign organisms/bodies, with no significant morphological differences observed either over time (day 2 vs. day 14) or relative to the culture medium from which  
Figure 7: Real-time trend, during Exp3, of the parameters pH, illumination, and temperature inside and outside the growth chambers. the microalgae derived their nutrients. Any irregularities within the algal biofilm depicted in Figure 8 are not due to the medium used, but to the drying phase of the biomass discs via lyophilisation, which can cause cracks as a result of the mechanical stress associated with the operating pressure [48]. Examining the cellular aggregates, it becomes apparent that cells seem interconnected by an external matrix (Figure 8). This morphological trait likely arises from the cells' capacity to secrete extracellular polymeric substances (EPSs) rich in polysaccharides [49], as confirmed by the results of the EDS investigations. Compositional analyses via EDS of the algal biofilm surface detected primarily carbon and oxygen, alongside traces of other elements naturally present in the culture media employed (for example sulphur, phosphorus, potassium, calcium, magnesium, and others). Complete EDS spectra of the analysed samples are provided in the Supplementary Materials. While the SEM characterisation in this study focused on the horizontal plane to examine the surface distribution of cells, future experimental investigations will focus on a more detailed study of the vertical section of the algal biofilm. This approach will provide more information on the porosity and morphology of the cells within the different photosynthetic layers, thus allowing further clarification of the structure and functions exhibited by the algal community. Regarding the second key objective of this study, namely the long-term biomass yields achievable by the system, this aspect was explored through statistical extrapolation rather than mechanistic modelling. Specifically, we applied simple predictive regression models to the temporal trends observed in the five experiments, as a means of statistical approximation. Table 4 presents the statistical parameters obtained for the three regression models tested--quadratic polynomial, three-parameter logistic, and two-parameter exponential--for the two conditions, A and W, as detailed in Section 2.6. The full statistical report of the models applied to the growth data is available in the Supplementary Materials. Although the quadratic polynomial regression exhibited the best statistical fit (Table 4), all three models produced similar performance metrics, suggesting high statistical reliability within the 15-day observation period. However, these models were applied purely as statistical tools for interpolation and extrapolation, rather than as biologically representative frameworks. As such, they do not capture the metabolic or physiological mechanisms governing the growth of _G. phlegrea_. Future studies aiming to better describe the system's long-term behaviour should consider incorporating biologically informed models that account for environmental variables and resource limitations.