Molecular Soil Biology 2026, Vol.17, No.1, 1-11 http://bioscipublisher.com/index.php/msb 9 4.2 Microalgal cultivation SA-2 was cultured in BBM supplemented with EDTA-Fe and EDTA-Ca at concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg/L. The pH of the BBM medium was adjusted to 8.0 using 1 mol/L NaOH. The culture conditions were maintained at (23±1) ℃, with a light intensity of 2 000 lx and a 16 h:8 h light/dark cycle. 4.3 Monitoring microalgal cell density Samples were collected every 24 h for optical density (OD) measurements. Prior to sampling, the culture was gently mixed to prevent cell sedimentation, then transferred to a cuvette. Absorbance was measured at 682 nm using a spectrophotometer. Each measurement was performed in triplicate and the mean value was calculated to reduce error. 4.4 Monitoring microalgal dry weight Microalgal cells were collected from the culture by centrifugation. The pellet was washed 2~3 times with distilled water, centrifuged again, and transferred to pre-weighed dry filter paper. The samples were dried in an oven at 60 ℃to constant weight. Dry weight was measured using an electronic balance. A dry weight standard curve was constructed based on OD values to convert measured OD values to microalgal dry weight. 4.5 Monitoring microalgal cell viability An appropriate volume of microalgal culture was centrifuged and the cells were stained with trypan blue for 3 min. The samples were decolorized overnight in chloral hydrate solution at room temperature, with the solution replaced 2~3 times. Finally, the samples were stored in 50% (v/v) glycerol and counted using a hemocytometer (Wang, 2020). 4.6 Determination of microalgal protein content Protein content was measured using a BCA assay kit (Qiu, 2021). Two milliliters of culture was centrifuged at 8 000 r for 8 min. The pellet was washed twice with deionized water and resuspended in 1 mol/L NaOH solution, boiled for 10 min, and centrifuged at 8 000 r for 8 min. The supernatant was collected, and the extraction process was repeated three times. Protein content in the algal cells was determined using the BCA assay kit. 4.7 Determination of microalgal lipid content Lipid content was determined using the vanillin–phosphoric acid colorimetric method (Mishra et al., 2014). One hundred microliters of algal culture were placed in a stoppered test tube, 2 mL concentrated sulfuric acid was added, and the mixture was heated in a 100 ℃water bath for 10 min. After cooling to room temperature in an ice bath, 5 mL of vanillin–phosphoric acid reagent was added. The reaction proceeded at 37 ℃and 200 rpm on a shaker for 15 min. Absorbance was measured at 530 nm, and lipid content was calculated using a standard curve. 4.8 Determination of microalgal pigment content Pigment content was determined by extraction (Abrha et al., 2025). Two milliliters of culture were centrifuged, the supernatant discarded, and the pellet washed twice with sterile water. Methanol was added, and the samples were extracted in the dark at 4 ℃for 12 h. Absorbance of the supernatant was measured at 750, 665, 652, and 480 nm using a spectrophotometer. Pigment concentrations were calculated using the corresponding formulas. 4.9 Determination of microalgal carbohydrate content Carbohydrate content was measured using the anthrone method (Brányiková et al., 2011). Microalgal cells were collected by centrifugation (3 000~5 000 r, 5~10 min) and washed with distilled water. Cells were disrupted by vortexing 0.5 mL glass beads in 0.25 mL distilled water for 4 min. To the pellet, 3.3 mL of 30% perchloric acid was added, stirred at 25 ℃for 15 min, and centrifuged to obtain the supernatant. This extraction was repeated three times, the extracts were combined, and the volume adjusted to 10 mL. A 0.5 mL aliquot of the extract was cooled to 0 ℃, mixed with 2.5 mL anthrone reagent, and heated in a 100 ℃water bath for 8 min. After cooling to 20 ℃, absorbance was measured at 625 nm. Carbohydrate content was calculated using a standard curve and a correction factor of 0.9.
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