## 2 Materials and Methods
### P. tricornutum Cultivation and Lipid Production
#### 2.1.3 Harvesting  
In this experiment, 100 L of culture was harvested using a disk stack centrifuge from the GEA Westfalia Separator GMBH model OSE 5-91-037. This equipment separates the liquids from the solids owing to the high speed (8000-9000 rpm) of the drum. The fresh microalgae paste is concentrated in the discs with a solid concentration between 15 and 17% depending on the concentration of the culture. * Growth parameters Optical density (OD) values were obtained using 2 mL of culture volume with a spectrophotometer (Biochrom Libra S11 Visible Spectrophotometer) at three different wavelengths (680, 668, and 750 nm). DW was measured using a 10 mL sample of culture filtered through previously dried glass fiber filters (47 mm PRAT DUMAS, France) and washed with 10 mL of distilled water for productivity analysis. Light (Light Meter HS1010A), pH, and temperature (ADWA AD11) measurements were also performed in this experiment. * Pigment analytical measurements Chlorophyll extraction and quantification were performed using ethanol as the organic solvent, as described by [29]. Five milliliters of the culture was centrifuged at 3000 rpm for 10 min. After discarding the supernatants, 5 mL of distilled water was added and centrifuged at 4500 rpm for 10 min. This rinse was repeated twice, and after discarding the supernatant, 5 mL 98% ethanol was added to the pellet. The tubes were stored in the absence of light at 6 \({}^{\circ}\)C for 24 h and then centrifuged at 4500 rpm for 10 min. Chlorophyll quantification was then performed spectrophotometrically using the supernatants and the following equation: \[\mu g_{\text{clorofia}}/\text{mL}_{\text{medium}}=(11.64\text{A}_{663}-2.16 \text{A}_{645}-0.10\text{A}_{630})\text{v}/(\text{IV}),\] (1) where A\({}_{\text{xxx}}\) is the absorbance at xxx nm after removing the sample absorbance at 750 nm, against a blank of the solvent used; \(\gamma\) means the volume of solvent used (mL); 1 is the spectrophotometric cell length (cm); and V is the sample volume (mL). Carotenoid extraction and quantification were performed using dimethyl sulfoxide (DMSO) as an organic solvent following the method described by [30], assuming fucoxanthin as the main carotenoid produced by _P. tricornutum_[31]. Fifty milliliters of the culture was centrifuged at 3600 rpm for 15 min. After discarding the supernatants, the pellets were stored in a freezer and dried via lyophilization. Five milliliters of DMSO was added to each 3.5 mg of biomass, homogenized and incubated at 70 \({}^{\circ}\)C for 5 min. The tubes were centrifuged at 4600 rpm for 10 min. Quantification of total carotenoids was performed spectrophotometrically using the supernatants and the following equation: \[\text{C}_{\text{Chl\,a}}\,(\text{mg}\cdot\text{L}^{-1})=13.34\text{A}_{666}-4. 85\text{A}_{650}\] (2) \[\text{C}_{\text{Chl\,b}}\,(\text{mg}\cdot\text{L}^{-1})=24.58\text{A}_{650}-6. 65\text{A}_{666}\] (3) Total car \[(\text{mg}\cdot\text{L}^{-1})=(1000\times\text{D}_{480}-1.29\times\text{C}_ {\text{Chl\,a}}-53.76\times\text{C}_{\text{Chl\,b}})/220\] (4)
* Lipid extraction Total lipid extractions were performed following the method described by [32], where 13.5 mL of chloroform, 6.5 mL of methanol, and 5.25 mL of distilled water (68% NaCl) were mixed with 1 g of dried biomass. The mixture was magnetically stirred for 10 min and then centrifuged at 3000 rpm for 10 min. The resultant organic lower phase was transferred to a clean glass vial and evaporated on a rotary evaporator to remove chloroform. The difference between the weight of the vial after evaporation and the weight of the empty vial was taken to be the weight of the lipids. The lipid content (% of dry weight) was the weight of lipids divided by the weight of the starting dry algal biomass (1 g). * Stress Experiments to study the effect of culture age were performed using three open raceways photobioreactors (RWP) inoculated with inoculum from a back-up system (RWP4). Two of them, RWP1 and RWP2 (R1 and R2) were inoculated again, while R3 was renewed.