## 2 Materials and Methods
### Biomass Characterization
#### 2.2.2 Determination of the Nutritional Composition of Microalgae Biomass  
The dry matter, moisture, ash, fat, protein, fiber, and carbohydrate contents and the energetic value of all microalgae biomass were determined.  
* Moisture content A sample of about 1 g was placed in a Petri dish inside an oven at 105 \({}^{\circ}\)C for 24 h and subsequently weighed [28]. Three replicates were performed. The moisture content was calculated through the following equation: Moisture content (%) = (weight at time 0 \(-\) weight of dried sample)/weight at time 0 \(\times\) 100 (3)
* Ash A sample of about 2.5 g was incenerated in a muffle furnace at 550 \(\pm\) 15 \({}^{\circ}\)C until constant weight [29]. The assay was performed in triplicate.
* Fat A sample of approximately 10 g was boiled in a beaker under reflux conditions with dilute hydrochloric acid (4 M) for 30 min, then filtered. The fat was extracted for 4 hours using petroleum ether in a Soxhlet extractor into a pre-weighed round-bottom flask. The solvent was evaporated using a rotary evaporator (Buchi R-210, Buchi Labortechnik AG, Flawil, Switzerland), and the residue was dried to a constant weight at 102 \(\pm\) 2 \({}^{\circ}\)C [29].
* Protein The protein content was determined according to ISO 1871:2009 [30]. Briefly, a sample of approximately 0.5 g was digested in a mineralization block (Kjeltec Foss) with concentrated sulfuric acid (96% \(w/w\)) in the presence of a catalyst. The nitrogen content (N) was calculated based on the quantity of ammonia produced. The protein content was then determined using the following formula: %protein = %N \(\times\) 6.25. A blank test was also conducted simultaneously. The assay was performed in triplicate.
* Fiber The methods used were based on AOAC 991.43 [31] and AOAC 985.29 [32], with the assay performed in triplicate. Approximately 1 g of sample was subjected to enzymatic digestion using three enzymes: \(\alpha\)-amylase (30 min at 90 \({}^{\circ}\)C), followed by protease (30 min at 60 \({}^{\circ}\)C), and amyloglucosidase (30 min at 60 \({}^{\circ}\)C). Insoluble fibers were separated from soluble fibers in the first filtration (using Celite). The residue was washed with 10 mL of water at 70 \({}^{\circ}\)C, followed by 10 mL of 95% ethanol (EtOH) and 10 mL of acetone. Fourvolumes of pre-heated EtOH (60 \({}^{\circ}\)C) were added to isolate the soluble fiber, and the residue was then washed with 15 mL each of 75% EtOH, 95% EtOH, and acetone. The residue was dried, and protein and ash contents were determined, with the remainder considered insoluble fiber, subtracting the weight of the Celite. Total fiber content was calculated by summing the insoluble and soluble fiber contents.
* Carbohydrates The carbohydrates were determined by difference according to the European Commission [33] and using the following formula: \[\text{Carbohydrates}=100-(\text{moisture}+\text{ash}+\text{fat}+\text{protein})\] (4)
* Energetic value For the determination of the energetic value, the following formula (Regulation UE n\({}^{\circ}\) 1169) [33] was used: \[\text{Energetic value (kcal}/100\text{ g DW)}=4\times(\text{mass}_{\text{protein}}+\text{ mass}_{\text{carbohydrates}})+9\times\text{mass}_{\text{fat}}\] (5)