### Rationale Behind the Selection of Twin-Layer Cultivation Technology  
The cultivation system implemented and used in this study is based on the operating principles of twin-layer type photobioreactors (TL-PBRs). These attached-biomass systems are based on the presence of two layers, namely, (i) a macroporous layer, which is traditionally referred to as the \"source layer\" and is the one through which the culture medium flows; and (ii) a microporous layer, which is conventionally referred to as the \"substrate layer\" and is the one onto which the biomass is attached [4; 21]. The first important advantage of this technology is the possibility offered by this system to safely use municipal wastewater as a microalgal growing medium at zero cost, which is an extremely advantageous condition from the point of view of process impacts. In fact, the presence of a microporous substrate guarantees that only the necessary nutrients for the organisms are able to pass through it, leaving the biomass isolated from the ensemble of microparticles and/or any micro-aggregates present in the municipal sewage, as well as competitor microorganisms [4]. As a result, the processed output should be a clean algal biomass devoid of extraneous aggregates that would invalidate its purity, which will provide market value benefits when produced for future applications in biotechnological sectors. This practical advantage becomes even more relevant and critical when dealing with primary effluents that have not yet undergone activated sludge oxidation treatment, as in the case of the present study (see Section 2.3). Such effluents are indeed full of potential because they are rich in their original nutrients that have not yet been removed/processed by activated sludge, but they also have higher turbidity levels due to the lack of treatment [24]. Because of this, when the intention is to cultivate microalgae in liquid phase PBR using municipal effluents (or other types of effluents), scientific evidences often confront stakeholders with a choice: (a) if the priority is to obtain higher-value clean biomass, then it shall be necessary to select less turbid (cleaner) effluents or to perform complex pre-treatments on the effluent before its contact with the biomass for cultivation, thus affecting the process performance [25]; (b) if the priority is achieving only a quantitatively high-performance cultivation, then it might be necessary to focus on other aspects of the cultivation system and this might affect the purity of the biomass and, consequently, also its sale value [18]. The system presented here makes it possible to avoid this choice and obtain a clean biomass with a low energy/material demand. The second important advantage offered by these systems is the possibility of cultivating the algal biomass in the form of a dense, low moisture algal paste, which can be easily harvested using spatulas (manually) or low-intensity air sprays (indirect harvesting). This last solution was proposed by [21], when hypothesising a method for more frequent and abundant harvesting on an industrial scale. Regarding this advantage, species belonging to the genus _Galdieria_ (as in this case) claim the advantage of typically developing and thriving in nature as algal mats on mineral substrates [26], thereby being able to (i) recover moisture and macro/micro nutrients through contact with the substrate on which they grow, and (ii) exchange gases directly through the interface in contact with the air. Consequently, the adoption of TL systems might prove to be a strategically successful choice for the cultivation of these valuable extremophilic microorganisms. Gathering the above considerations, the last important advantage consists of the overall energy efficiency of the system.