## 2 Materials and Methods
### Outdoor Photo-Bioreactors Cultivation of Microalgae on the Best Dilution Ratio  
The best dilution ratio (1:50) obtained in the first screening tests was further applied for microalgae cultivation in outdoor condition to be the most representative of further industrial-scale. For this purpose, _Monoraploidium_ sp. was cultivated outdoors, between August and November 2019, in a 110 dm\({}^{3}\) (0.29 m \(\times\) 1.65 m) vertical closed photobioreactors with an active volume of 73 dm\({}^{3}\) located in Lescar (France) (Figure 1 and Figure S2). Tap water was used to formulate the culture medium, where ALD was used as nutrients source at a dilution ratio of 1:50. The first batch started with an initial algae biomass concentration of 0.12 gTSS/L. The culture was bubbled with air and the pH maintained within a desired range (pH 7.3-8.1, with a set point of 7.7) to support algae growth. The pH was controlled by a pH meter and was regulated by CO\({}_{2}\) injection in the system through a solenoid valve. When the pH reached the set point (7.7), the controller opened the valve to allow CO\({}_{2}\) injection, so that CO\({}_{2}\) reduces pH level.  
First, three experiments in batch mode were carried out in order to optimize the photo-bioreactor performance and to define the optimal retention time. Once the system was optimized, the culture was switched to semi-continuous mode which lasted from September to November 2019. The retention time applied was 15 days and the feeding of ALD was performed using a peristatic pump (Figure 1). Climatological data in Lescar region were extracted from the website of \"Meteo France\" ([[http://www.meteofrance.com/accueil](http://www.meteofrance.com/accueil)]([http://www.meteofrance.com/accueil](http://www.meteofrance.com/accueil))).  
Microalgae growth was daily monitored by determining Optical Density (OD) at 680 nm with a UV-visible spectrometer (7315, Jenway, United Kingdom), after proper dilution with deionized water to obtain absorbance values between zero and one. In parallel, the microalgae growth was measured by counting cells system using Malassez method ([PERSON] 1973, [PERSON](r), Germany). Briefly, Malassez method consists of a thick glass microscope slide (Malassez) with a rectangular indentation  
Figure 1: Scheme of experimental set up for microalgae production in semi-continuous mode in outdoor conditions in the vertical closed photobioreactors.  
that creates a chamber of a well-known volume, so the number of contained cells can be determined. Light microscopy was carried out using a Zeiss Axioskop 2 equipped with an AxioCam MRc camera.  
Total suspended solids (TSS) were measured by filtering 5-15 mL culture solution through a glass fiber filter (Whatman GF/A). TSS was determined gravimetrically as a difference of the filters before and after drying at 105 \({}^{\circ}\)C for 24 h.  
The microalgae biomass was harvested during the semi-continuous phase by drying it at 70 \({}^{\circ}\)C, and it was then conserved at room temperature in a dry container. The Equation (3) was applied in order to calculate the biomass productivity (P):  
\[P=(DW_{i}-DW_{0})/(t_{i}-t_{0}) \tag{3}\]  
where \(DW_{i}\) and \(DW_{0}\) are the Dry Weights (gTSS L\({}^{-1}\)) at time \(t_{i}\) and \(t_{0}\) (initial time), respectively.