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Appl. Sci. 2020, 10, 2531 4 of 18 2.3. Cell Growth and Biomass Analysis Wild-type P. tricornutum was cultured in 5 mL of L1 medium in a flat-bottomed sterile 6-well culture plate with a lid (Costar®, Cat no. 3516). The cultures were maintained with consistent shaking on an orbital shaker at 130 rpm. Cell growth was monitored by measuring the optical density at 680 nm using a Synergy H1 Microplate Reader, BioTek. All the conditions were studied in triplicate. The biomass was estimated by dry weight from the centrifuging of P. tricornutum cultures at 7000× g for 10 min on the 10th day. The supernatant was discarded, and the pellet was dried at 60 ◦C for biomass analysis until the constant mass was achieved. 2.4. Visualization of Lipid Bodies Intracellular lipid bodies (LBs) were visualized using a modified Nile Red (9-diethylamino-5H-benzo[a]-phenoxazine-5-one) staining method [17,18]. Briefly, 1 mL of the algal culture was centrifuged at 10,625× g for 10 min. Then, the cell pellet was resuspended in 500 μL of 20% dimethyl sulfoxide (DMSO) and vortexed for 1 min at room temperature. Cells were centrifuged at 10,625× g for 5 min, and the cell pellet was resuspended in 500 μL of water and vortexed before adding Nile red solution (0.5 mg mL−1 dissolved in acetone) and incubated for 5 min in dark at room temperature. Stained LBs imaging was performed under a confocal laser scanning microscope (Leica SP8) using 40× magnification. The Nile red fluorescence was detected using a UV light source at excitation/emission wavelengths of 488 and (490–550) nm. 2.5. Quantification of Total Lipids Total lipids were extracted using Bligh and Dyer method [19] with the following modifications. To a 5 mL Eppendorf tube containing a known amount of dry algal biomass, mixtures of methanol and chloroform were added in 2:1 ratio. The mixture was vortexed for 2 min and incubated at room temperature for 24 h, after which 1 mL of chloroform and 0.9 mL of water were added. The mixture was vortexed for 2 min, and the different layers were separated by centrifugation for 10 min at 300× g. The lower layer was evaporated, and the residue was dried at 80 ◦C for 30 min. The weight was calculated using a precision scale. 2.6. Nile Red Screening In order to detect neutral lipids, Nile red assay [20,21] was done in 96-well plate. Briefly, 250 μL of culture was added with 15 μL of Nile red dissolved in acetone from the stock solution of 0.5 mg mL−1. The plate was incubated in the dark at room temperature for 30 min. The fluorescence intensity was measured at an excitation of 530 nm and emission of 590 nm using a Synergy H1 Microplate Reader, BioTek. 2.7. Fatty Acid Profiling Fatty acid methyl esters were prepared directly from the wet algal biomass. NaOH in methanol (1 mL of 0.5 N) was added to test tubes containing the biomass; then, the tubes were placed in a sonicating bath for 3 min, followed by heating at 90 ◦C for 10 min. The samples were allowed to cool, and 1 mL of 1.5% H2SO4 in methanol was added. Then, samples were heated again at 90 ◦C for 10 min. After cooling, 1 mL of water and 1 mL of hexane were added. Test tubes were vortexed for 2 min and then centrifuged at 1200× g for 5 min. The hexane layer containing Fatty acid methyl esters analysis (FAME) was recovered and dried over anhydrous sodium sulfate. FAME were quantified using temperature-programmed gas liquid chromatography on a Scion 436 gas chromatograph fitted with a 30 m × 0.25 mm column coated with 50% cyanopropyl-methylpolysiloxane (DB-23) and linked to a computerized integration system [22]. The fatty acid data were expressed as the mass percent of total fatty acid identified. Furthermore, the fatty acid profile was used to analyze the physical properties of biodiesel using biodiesel analyzer [21,23,24].PDF Image | Red Light Variation Lipid Profiles in Phaeodactylum tricornutum
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