1 Introduction

Against the backdrop of global ecological change and increasing resource demands, microalgae have received widespread attention as a biomass resource with tremendous potential. Microalgae are a group of algae that are widely distributed on land and in aquatic environments, microscopic in size, and whose morphology can only be distinguished under a microscope. They can utilize light, carbon dioxide, and water to carry out photosynthesis, efficiently producing a variety of functional bioactive substances, including proteins, polysaccharides, lipids, and pigments.Microalgae are capable of growing in diverse environments and exhibit rapid growth rates and high biomass accumulation. Owing to their rich nutritional composition and diverse functional components, microalgae show broad application prospects in the field of biological resource development (Fabris et al., 2020).

As an important source of natural compounds, microalgae not only efficiently synthesize biofuels and polysaccharides but also possess the potential for producing high-value-added products. Pigments, essential amino acids, and vitamins contained in their cellular matrix form the basis for their application in the food industry, while their abundance of long-chain polyunsaturated fatty acids enhances their value in the development of nutritional supplements (Markou and Nerantzis, 2013).These multidimensional utilization characteristics make microalgae a strategically important raw material in sustainable biomanufacturing systems (Shin et al., 2015).

EDTA is a widely used chelating agent that forms stable complexes with heavy metals, thereby reducing the toxic effects of heavy metals on plants. Owing to its chelating properties, EDTA is extensively applied in industries such as textiles, papermaking, food processing, medicine, and agriculture for purposes including water softening, boiler descaling, metal de-rusting, electroplating, and the enhanced remediation of heavy-metal-contaminated soils.However, the widespread use of EDTA has led to its significant accumulation in the environment, posing potential ecological risks. Although EDTA itself is non-toxic at low concentrations, its chemical stability and resistance to biodegradation enable its long-term persistence and accumulation in the environment. Meanwhile, the prolonged environmental presence of EDTA can chelate toxic heavy metal ions already deposited in sediments, allowing these ions to re-enter aquatic systems and migrate freely, leading to environmental pollution (Claudia and Jaime, 2003) and disrupting the balance of essential nutrients required for algal growth.

For example, metal ions such as iron, zinc, and magnesium are essential micronutrients in the photosynthetic and metabolic processes of algae. Excessive EDTA reduces the availability of these ions, thereby inhibiting normal algal growth and reproduction, slowing growth rates, and reducing cell division. EDTA also affects essential metabolic processes, including photosynthesis and respiration. It may interfere with chlorophyll synthesis and function, reduce photosynthetic efficiency, and limit the ability of algae to acquire energy, ultimately impairing growth and development. For instance, when the EDTA concentration exceeds 13.5 μmol/L, it significantly inhibits the growth of Microcystis aeruginosa (Chu et al., 2007). At low concentrations, however, EDTA can promote microalgal growth and biomass formation. Some studies have shown that low concentrations of EDTA can significantly alter metal toxicity to algae through chelation (Fawaz et al., 2018). When metals form stable complexes with EDTA, their bioavailability is markedly reduced, primarily through decreased concentrations of free metal ions in water, which are the most biologically accessible forms. This chelating mechanism diminishes the biological toxicity of metals, leading to a substantial reduction in toxicity indicators. Such phenomena have been confirmed in multiple toxicological studies involving metal–EDTA complex systems, verifying that the formation of metal–EDTA complexes is the primary mechanism for toxicity reduction (Geis et al., 2000). Some studies have found that high concentrations of EDTA strongly inhibit the growth of Microcystis aeruginosa but have little effect on Scenedesmus quadricauda (Zeng et al., 2009). Other reports indicate that EDTA supplementation can enhance lipid accumulation in microalgae (Ren et al., 2014). For instance, in Nannochloropsis oculata, both biomass and lipid accumulation increase progressively with rising EDTA concentrations (Xiao et al., 2013).

Iron is one of the most important trace mineral elements in living organisms and is an essential component of intracellular redox reactions. It plays critical roles in cellular respiration, photosynthesis, and catalytic reactions involving metalloproteins. As an indispensable micronutrient for the growth and development of photosynthetic organisms, iron’s metabolic functions are mainly reflected in the molecular regulation of enzyme cofactors. Its transition metal properties provide a central role in electron transport chains and redox-catalyzed reactions, participating in metabolism through diverse structural forms, including iron–sulfur clusters, heme, di-iron centers, and mononuclear iron. In higher plants and microalgae, the iron–sulfur cluster biosynthesis systems—evolved from endosymbiotic bacteria—are located in the mitochondria and chloroplasts. Their precise assembly mechanisms support the continuous cofactor demand required for photosynthesis, respiration, and other energy metabolism processes (Balk and Schaedler, 2014). Iron deficiency disrupts electron transport and reduces energy conversion efficiency. Moreover, iron is an essential cofactor for enzymes such as RuBisCO and catalase, participating in carbon fixation and reactive oxygen species detoxification. Iron is also a key element in chlorophyll synthesis, the photosynthetic electron transport chain (e.g., cytochromes, ferredoxin), and enzymatic activities (e.g., catalase). Thus, it plays crucial roles in microalgal growth, metabolism, and biomass formation. Supplementing iron during the nutrient phase positively influences the photosynthetic mechanisms of the microalgae strain SA-2. Under mixotrophic conditions, iron significantly affects biomass, chlorophyll, carbohydrate, protein, and lipid synthesis in microalgae (Xia et al., 2010). Furthermore, relevant studies show that iron plays an essential role in microalgal growth and lipid accumulation. At certain concentrations, iron ions influence biomass, lipid composition, and metabolite synthesis, with high iron concentrations significantly affecting oleic acid accumulation (Zhang et al., 2014).

Calcium is recognized as the second essential nutrient element in plants and is an important component of cell membranes. It affects the middle lamella of cell walls and plays a vital role in cell division, growth, and death (Lei et al., 2012). Calcium ions also significantly influence carbohydrate formation and transformation.

Some studies indicate that calcium ions promote lipid synthesis in microalgae, and their oxides can catalyze microalgal oil synthesis for biodiesel production (Chen et al., 2016). At low concentrations, calcium ions promote microalgal growth, increase biomass, and enhance lipid accumulation, although the effects are modest (Gao, 2024). High concentrations of calcium ions also promote biofilm formation derived from algal organic matter in the microalgal strain SA-2 (Fan et al., 2025).Currently, most studies regarding microalgae and calcium focus on the role of microalgae in balancing environmental calcium ions, with calcium concentrations negatively correlated with the growth of calcified microalgae (Zhao et al., 2020). Research on the effects of calcium on microalgal growth and biomass accumulation remains limited.However, in certain plants—such as Brassica campestris—supplementing calcium under stress conditions promotes growth and photosynthesis. Foliar calcium application increases flavonoid content, enhances electron transport rates, alleviates photosynthetic inhibition, and improves photosynthetic efficiency. Calcium ions also mitigate excessive acidification of the thylakoid membrane, maintain membrane integrity, and enhance ATPase activity (Cheng, 2020).As calcium ions support thylakoid membrane structure and the oxygen-evolving complex in PSII, appropriate concentrations of calcium may similarly promote microalgal growth and biomass synthesis.

Excessive accumulation of EDTA-Ca and EDTA-Fe can elevate calcium and iron concentrations in the environment, potentially causing heavy metal-like stress and impairing normal microalgal growth. However, studies indicate that the exogenous addition of EDTA-Fe and EDTA-Ca complexes can significantly regulate the photosynthetic metabolic system of the microalgae SA-2 under mixotrophic conditions. Iron sources not only enhance biomass production by improving light energy conversion efficiency but also exhibit dose-dependent effects on intracellular chlorophyll, carbohydrate, protein, and macromolecular synthesis. Particularly in lipid metabolic regulation, EDTA-Fe displays concentration-dependent modulation, with oleic acid showing specific enrichment in triacylglycerols when the concentration reaches a threshold level (Kona et al., 2017).

2 Results and Analysis

This study aims to investigate in depth the variation patterns and influencing factors of microalgal biomass content under EDTA-Fe and EDTA-Ca treatments. The expected outcomes will provide a theoretical basis for the efficient cultivation of microalgae and the optimized utilization of biomass resources, as well as technical support for further research on biomass accumulation in microalgae. Experimental results showed that supplementing the nutrient phase of microalgae SA-2 with EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L could promote the growth of SA-2 to a certain extent and increase its biomass yield. The optimal concentration for EDTA-Ca to promote SA-2 growth was 0.3 mg/L, whereas that for EDTA-Fe was 0.2 mg/L. At their respective optimal concentrations, both EDTA-Ca and EDTA-Fe enhanced cell density, dry weight, cell viability, and the contents of proteins, lipids, pigments, and carbohydrates in SA-2. The addition of EDTA-Ca and EDTA-Fe did not alter the logarithmic growth phase of SA-2, which was consistent with that of the untreated control. The promotive effect of EDTA-Fe at its optimal concentration on the growth and biomass synthesis of SA-2 was greater than that of EDTA-Ca at its optimal concentration. This indicates that iron, compared with calcium, plays a more critical role in the growth, reproduction, and biomass formation of microalgae, and that microalgae have a higher demand for iron. Furthermore, the optimal concentration of EDTA-Fe for SA-2 growth was lower than that of EDTA-Ca, suggesting that microalgae are more sensitive to iron than to calcium and are more susceptible to stress caused by high concentrations of iron ions.

2.1 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal cell density

Microalgae SA-2 exhibited the highest cell density and best growth performance when the concentration of EDTA-Ca was 0.3 mg/L. Concentrations of EDTA-Ca above 0.3 mg/L showed an inhibitory effect on SA-2 cell density (Figure 1a). For EDTA-Fe, the optimal concentration for promoting SA-2 growth was 0.2 mg/L, whereas concentrations above 0.2 mg/L began to inhibit microalgal growth (Figure 1b). These results indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L promoted the growth of SA-2, while higher concentrations of EDTA-Ca (>0.3 mg/L) and EDTA-Fe (>0.2 mg/L) inhibited growth. The cell density of SA-2 increased exponentially between days 9 and 10 (Figure 1), indicating that day 10 corresponds to the logarithmic growth phase. The addition of EDTA-Ca and EDTA-Fe did not significantly affect the duration or timing of the logarithmic growth phase of SA-2.

Figure 1 Changes in cell density of microalga SA-2 under different treatments Note: (a) EDTA-Ca at different concentrations; (b) EDTA-Fe at different concentrations

2.2 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal dry weight

The dry weight of microalgae SA-2 was calculated based on the constructed dry weight standard curve (Figure 2a). Changes in dry weight were consistent with changes in cell density. The maximum dry weight of SA-2 was observed at an EDTA-Ca concentration of 0.3 mg/L and an EDTA-Fe concentration of 0.2 mg/L, both reaching 0.9 g/L (Figure 2b; Figure 2c). These results indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L promoted the growth of microalgae. In contrast, EDTA-Ca concentrations above 0.3 mg/L and EDTA-Fe concentrations above 0.2 mg/L inhibited microalgal growth.

Figure 2 Changes in dry weight of microalga SA-2 under different treatments Note: (a) Dry weight standard curve; (b) EDTA-Ca at different concentrations; (c) EDTA-Fe at different concentrations

2.3 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal cell viability

In this study, the effects of EDTA-Ca and EDTA-Fe treatments on microalgal cell viability were measured on day 12, and micrographs of SA-2 cells under different EDTA-Ca and EDTA-Fe concentration gradients were obtained (Figure 3). The strongest cell viability (96.50%) was observed under 0.3 mg/L EDTA-Ca treatment (Figure 4a), while the highest viability (97.24%) occurred under 0.2 mg/L EDTA-Fe treatment (Figure 4b). These results indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L enhance the cell viability of SA-2. Conversely, EDTA-Ca concentrations above 0.3 mg/L and EDTA-Fe concentrations above 0.2 mg/L inhibit cell viability, leading to increased microalgal cell death.

Figure 3 Microscopic images of microalga SA-2 cells under different concentrations of EDTA-Ca and EDTA-Fe treatments

Figure 4 Changes in cell viability of microalga SA-2 under different treatments Note: (a) EDTA-Ca at different concentrations; (b) EDTA-Fe at different concentrations. Different lowercase letters indicate a significant difference (P<0.05)

2.4 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal protein content

Changes in microalgal protein content on day 12 were measured under different concentrations of EDTA-Ca and EDTA-Fe. The results showed that protein content was highest at an EDTA-Ca concentration of 0.3 mg/L, reaching 0.1 mg/mL (Figure 5a). For EDTA-Fe, the maximum protein synthesis occurred at 0.2 mg/L, with a protein content of 0.14 mg/mL (Figure 5b). These findings indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L promoted protein synthesis in SA-2. Conversely, EDTA-Ca concentrations above 0.3 mg/L and EDTA-Fe concentrations above 0.2 mg/L inhibited protein synthesis. The promotive effect of low-concentration EDTA-Fe (<0.2 mg/L) on protein synthesis was more pronounced than that of low-concentration EDTA-Ca (<0.3 mg/L), whereas the inhibitory effect of high-concentration EDTA-Fe (>0.2 mg/L) was stronger than that of high-concentration EDTA-Ca (>0.3 mg/L).

Figure 5 Changes in protein content of microalgae SA-2 Note: (a) treatment groups with different concentrations of EDTA-Ca; (b) treatment groups with different concentrations of EDTA-Fe. Different lowercase letters indicate a significant difference (P<0.05)

2.5 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal lipid content

The changes in microalgal lipid content under different concentrations of EDTA-Ca and EDTA-Fe were measured. Lipid content reached its maximum under 0.3 mg/L EDTA-Ca treatment, with a value of 11.83 mg/L (Figure 6b). For EDTA-Fe, the highest lipid content was observed at 0.2 mg/L, reaching 13.45 mg/L (Figure 6c). These results indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L promoted lipid synthesis in SA-2. Conversely, EDTA-Ca concentrations above 0.3 mg/L and EDTA-Fe concentrations above 0.2 mg/L inhibited lipid synthesis. The promotive effect of low-concentration EDTA-Fe (<0.2 mg/L) on lipid accumulation was more pronounced than that of low-concentration EDTA-Ca (<0.3 mg/L), while the inhibitory effect of high-concentration EDTA-Fe (>0.2 mg/L) was stronger than that of high-concentration EDTA-Ca (>0.3 mg/L).

Figure 6 Changes in lipid content of microalgae SA-2 Note: (a) Standard curve of lipid content; (b)treatment groups with different concentrations of EDTA-Ca; (c) treatment groups with different concentrations of EDTA-Fe. Different lowercase letters indicate a significant difference (P<0.05)

2.6 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal pigment content

Pigment content of microalgae under different concentrations of EDTA-Ca and EDTA-Fe was measured at various wavelengths. Total chlorophyll content reached its maximum under 0.3 mg/L EDTA-Ca, at 12.24 mg/L (Figure 7a), and under 0.2 mg/L EDTA-Fe, at 10.19 mg/L (Figure 7b). Chlorophyll a content peaked at 10.60 mg/L with 0.3 mg/L EDTA-Ca (Figure 7c) and at 16.34 mg/L with 0.2 mg/L EDTA-Fe (Figure 7d). Chlorophyll b content was highest at 2.74 mg/L under 0.3 mg/L EDTA-Ca (Figure 7e) and 13.32 mg/L under 0.2 mg/L EDTA-Fe (Figure 7f). Carotenoid content reached a maximum of 2.74 mg/L under 0.3 mg/L EDTA-Ca (Figure 7g) and 4.47 mg/L under 0.2 mg/L EDTA-Fe (Figure 7h).These results indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L promoted the synthesis of various pigments in SA-2, whereas higher concentrations of EDTA-Ca (>0.3 mg/L) and EDTA-Fe (>0.2 mg/L) inhibited pigment synthesis. The promotive effect of low-concentration EDTA-Fe (<0.2 mg/L) was more pronounced than that of low-concentration EDTA-Ca (<0.3 mg/L), while the inhibitory effect of high-concentration EDTA-Fe (>0.2 mg/L) was stronger than that of high-concentration EDTA-Ca (>0.3 mg/L). This difference may be attributed to the role of iron in multiple aspects of microalgal physiology, including photosynthetic electron transport, chloroplast development, key enzyme activities, and photoprotective mechanisms, suggesting that iron is more critical than calcium for chlorophyll synthesis in microalgae.

Figure 7 Changes in Pigment Content of Microalga SA-2 Note: (a) Total pigment content of microalgae SA-2 under different EDTA-Ca concentrations; (b) Total pigment content of microalgae SA-2 under different EDTA-Fe concentrations; (c) Chlorophyll a content of microalgae SA-2 under different EDTA-Ca concentrations; (d) Chlorophyll a content of microalgae SA-2 under different EDTA-Fe concentrations; (e) Chlorophyll b content of microalgae SA-2 under different EDTA-Ca concentrations; (f) Chlorophyll b content of microalgae SA-2 under different EDTA-Fe concentrations; (g) Carotenoid content of microalgae SA-2 under different EDTA-Ca concentrations; (h) Carotenoid content of microalgae SA-2 under different EDTA-Fe concentrations. Different lowercase letters indicate a significant difference (P<0.05)

2.7 Effects of EDTA-Fe and EDTA-Ca treatments on microalgal carbohydrate content

Changes in microalgal carbohydrate content on day 12 were measured under different concentrations of EDTA-Ca and EDTA-Fe (Figure 8). Carbohydrate content reached its maximum at 58.13 mg/L under 0.3 mg/L EDTA-Ca. For EDTA-Fe, the highest carbohydrate content of 80.04 mg/L was observed at 0.2 mg/L. These results indicate that EDTA-Ca at concentrations below 0.3 mg/L and EDTA-Fe at concentrations below 0.2 mg/L promoted carbohydrate synthesis in SA-2. Conversely, EDTA-Ca concentrations above 0.3 mg/L and EDTA-Fe concentrations above 0.2 mg/L inhibited carbohydrate accumulation in the microalgae.

Figure 8 Changes in carbohydrate content of microalgae SA-2 Note: (a) Standard curve for carbohydrate content; (b) treatment groups with different concentrations of EDTA-Ca; (c) treatment groups with different concentrations of EDTA-Fe. Different lowercase letters indicate a significant difference(P<0.05)

3 Discussion

In this study, EDTA-Fe and EDTA-Ca exhibited differential effects on microalgal biomass accumulation. The results indicate that supplementation with appropriate concentrations of EDTA-Fe and EDTA-Ca generally enhanced biomass content, whereas excessive concentrations of either EDTA-Fe or EDTA-Ca exerted inhibitory effects on SA-2. This observation is closely related to the distinct roles of the two elements in microalgal metabolism. Iron ions function as cofactors in enzymatic reactions, particularly those involving iron-containing proteins, and participate in the photosynthetic electron transport chain. Microalgae are rich in iron-binding proteins such as iron–sulfur (Fe-S) proteins, ribonucleotide reductase (RNR), and hemoproteins, with iron serving as a cofactor in DNA replication, DNA repair, cell cycle progression, metabolic catalysis, and iron homeostasis (Zhang, 2014). Iron is also a core element for photosynthetic electron transport, chlorophyll synthesis, and multiple enzymatic reactions, with its sufficient supply directly determining photosynthetic efficiency and organic carbon fixation rates.In contrast, calcium primarily contributes to cellular structure, signal transduction, and physiological regulation rather than directly participating in photosynthesis. Unlike nitrogen or phosphorus, it does not directly constitute biomass (e.g., proteins or nucleic acids), nor does it directly drive photosynthetic electron transport like iron. Its role is to provide “support” and “stability” within the cell (Thomas et al., 2023), and a stable intracellular environment is a prerequisite for efficient metabolic activities, including protein, lipid, and carbohydrate synthesis. The results of this study are consistent with previous reports highlighting the growth-promoting effects of iron on microalgae.

The chelating effect of EDTA plays a key role in microalgal cultivation. By binding Fe³⁺ and Ca²⁺, EDTA prevents the rapid precipitation of these metal ions in the medium, thereby maintaining their bioavailability and extending their effective period in the culture. EDTA not only facilitates the uptake of metal ions but also indirectly regulates metabolic processes, improving light energy utilization and carbon flux allocation. Chelation of Fe by EDTA ensures the normal synthesis of key cellular components, such as iron–sulfur proteins and cytochromes, enhancing electron transport efficiency and promoting accumulation of photosynthetic products. Proper levels of EDTA-Ca help stabilize cell structure and regulate transmembrane ion channels, thereby improving overall cellular homeostasis. These positive effects synergistically enable higher growth rates and biomass production, with the presence of EDTA being a critical prerequisite for the promotive effects of both elements. EDTA’s chelation satisfies the continuous demand for metal ions during metabolic processes.

Microalgae, as microorganisms with high light energy utilization efficiency and short growth cycles (Zhou and Ruan, 2014), hold significant potential in sustainable energy and biofuel production, the production of high-value nutrients and food additives, environmental remediation and carbon neutrality, as well as agriculture and fertilizer applications. Their further potential remains to be explored. This study systematically investigated the differential effects of EDTA-chelated metal ions (Fe and Ca) on microalgal biomass accumulation, providing new insights into strategies for enhancing biomass production. By referring to domestic and international reports on microalgal physiological responses to stress and the role of metal chelators in plants and algae, this study analyzed the effects of EDTA-Fe and EDTA-Ca on microalgal growth characteristics, cell viability, and biomass components including proteins, lipids, pigments, and carbohydrates. It was found that appropriate concentrations of EDTA-Fe and EDTA-Ca promoted the accumulation of various biomasses and cell biomass in SA-2, whereas excessive concentrations inhibited both biomass content and cell growth. These findings provide a theoretical basis for effective micronutrient supplementation strategies in microalgal cultivation.

Future studies could further explore the synergistic effects of combined EDTA-Fe and EDTA-Ca supplementation, and, based on the specific metabolic characteristics of target algal species, develop customized chelated micronutrient nutrition schemes. Such approaches could enable integrated strategies for EDTA-Fe and EDTA-Ca supplementation in large-scale algal cultivation systems, improving biomass yield and the concurrent accumulation of high-value products such as lipids, polysaccharides, or pigments. With further process optimization and ecological safety evaluation, these findings are expected to advance the application of microalgae in biofuels, carbon mitigation, and bioproduct development.

4 Materials and Methods

4.1 Materials

The microalga Nannochloris sp. SA-2 used in this study was isolated from saline-alkaline soil in Anda City, Heilongjiang Province, and was previously identified and preserved in our laboratory. It was cultured in Bold’s Basal Medium (BBM) under shaking conditions at 100 r/min. The cultivation temperature was maintained at (23±1) ℃, with a light/dark photoperiod of 16 h:8 h and a light intensity of 2 000 lx.

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.

Acknowledgments

This study was supported jointly by the Ministry of Education Innovation Team Science Fund Project (IRT_17R99) and the Heilongjiang Province College Student Innovation and Entrepreneurship Training Program Project (202410225509).

Conflict of Interest Disclosure

The authors declare no competing interests.

References

Abrha G.T., Makaranga A., and Jutur P.P., 2025, Enhanced lipid accumulation in microalgae Scenedesmus sp. under nitrogen limitation, Enzyme and Microbial Technology, 182: 110546.

https://doi.org/10.1016/j.enzmictec.2024.110546

Balk J., and Schaedler A.T., 2014, Iron cofactor assembly in plants, Annual Review of Plant Biology, 65(1): 125-153.

https://doi.org/10.1146/annurev-arplant-050213-035759

Brányiková I., Maršálková B., Doucha J., Brányik T., Bišová K., Zachleder V., and Vítová M., 2011, Microalgae-novel highly efficient starch producers, Biotechnology and Bioengineering, 108(4): 766-776.

https://doi.org/10.1002/bit.23016

Chen Y., Zhu G., Sun Y.J., 2016, Biodiesel production from microalgal lipids catalyzed by Ca-Mg-Al composite metal oxides, Journal of Qingdao University of Science and Technology (Natural Science Edition), 37(3): 260-264.

Cheng X., 2020, Response of photosynthesis in Chinese cabbage to rhizosphere phosphorus supply and regulation by calcium, Master's thesis, Shenyang Agricultural University, Advisor: Liu Y.F., pp.1-120.

Chu Z.S., Jin X.C., Yan F., Zheng S.F., Pang Y., and Zeng Q.R., 2007, Effects of EDTA and iron on the growth and competition of Microcystis aeruginosa and Scenedesmus quadricauda, Environmental Science, (11): 2457-2461.

Claudia O., and Jaime R., 2003, EDTA: the chelating agent under environmental scrutiny, Química Nova, 26(6): 901-907.

https://doi.org/10.1590/S0100-40422003000600020

Fabris M., Abbriano R.M., Pernice M., Sutherland D.L., Commault A.S., Hall C.C., Labeeuw L., McCauley J., Kuzhiumparambil U., Chen H., Ralph P.J., 2020, Emerging technologies in algal biotechnology: toward the establishment of a sustainable, algae-based bioeconomy, Front. Plant Sci., 11(1): 279.

https://doi.org/10.3389/fpls.2020.00279

Fan Y.C., Liu X.Y., Ni M.F., Wang Z.H., Zhang R.Y., Luo X.Y., Wang Z.K., 2025, Effect of calcium and magnesium on adsorption characteristics of extracellular polymeric substances for algal-derived organic matter, Chinese Journal of Environmental Science, 45(7): 3896-3904.

Fawaz G.E., Salam A.D., Kamareddine L., 2018, Evaluation of copper toxicity using site specific algae and water chemistry: Field validation of laboratory bioassays, Ecotoxicology and Environmental Safety, 155: 59-65.

https://doi.org/10.1016/j.ecoenv.2018.02.054

Gao L.L., 2024, Study on the effect of light and Ca²⁺/Mg²⁺ on algal-bacterial granular sludge, Master's thesis, Yangzhou University, Advisors: He Chengda, Huo Xiubing, pp.1-100.

Geis S.W., Fleming K.L., Korthals E.T., Searle G., Reynolds L., and Karner D.A., 2000, Modifications to the algal growth inhibition test for use as a regulatory assay, Environmental Toxicology and Chemistry, 19(1): 36-41.

https://doi.org/10.1002/etc.5620190105

Kona R., Hemalatha M., Srivastav K.V., and Venkata Mohan S., 2017, Regulatory effect of Fe-EDTA on mixotrophic cultivation of Chlorella sp. towards biomass growth and metabolite production, Bioresource Technology, 244(P2): 1227-1234.

https://doi.org/10.1016/j.biortech.2017.06.028

Lei J.C., Li Z.J., Zhou J., Dai H.L., 2012, Application of active dry yeast in soy sauce, China Brewing, 31(6): 162-165.

Markou G., and Nerantzis E., 2013, Microalgae for high-value compounds and biofuels production: A review with focus on cultivation under stress conditions, Biotechnology Advances, 31(8): 1532-1542.

https://doi.org/10.1016/j.biotechadv.2013.07.011

Mishra S.K., Suh W.I., Farooq W., Moon M., Shrivastav A., Park M.S., and Yang J.W., 2014, Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method, Bioresource Technology, 155: 330-333.

https://doi.org/10.1016/j.biortech.2013.12.077

Qiu W.S., 2021, Cultivation of Chlorella and co-production of lipids and paramylon, Master's thesis, Fujian Normal University, Advisor: Chen B.L., pp.1-110.

Ren H.Y., Liu B.F., Kong F., Zhao L., Xie G.J., and Ren N.Q., 2014, Enhanced lipid accumulation of green microalga Scenedesmus sp. by metal ions and EDTA addition, Bioresource Technology, 169: 763-767.

https://doi.org/10.1016/j.biortech.2014.06.062

Shin D.Y., Cho H.U., Utomo J.C., Choi Y.N., Xu X., and Park J.M., 2015, Biodiesel production from Scenedesmus bijuga grown in anaerobically digested food wastewater effluent, Bioresource Technology, 184: 215-221.

https://doi.org/10.1016/j.biortech.2014.10.090

Thomas N.L., Dart C., and Helassa N., 2023, Editorial: The role of calcium and calcium binding proteins in cell physiology and disease, Frontiers in Physiology, 14: 1228885.

https://doi.org/10.3389/fphys.2023.1228885

Wang M., 2020, Growth, lipid accumulation, and transcriptomic dynamics of Nannochloris sp. JB17 under carbonate stress, Ph.D. thesis, Northeast Forestry University, Advisors: Liu C.K., Bu Y.Y., pp.9-10.

Xia J.L., Li L., Wan M.X., Liu P., Wang R.M., Huang B., and Qiu G.Z., 2010, Isolation and identification of two microalgae strains and the effect of Fe³⁺ on their growth and lipid accumulation, Journal of Wuhan University (Natural Sciences Edition), 56(3): 325-330.

Xiao D., Lux X.H., Lu M.Z., Yu L.S., Xue R., and Jian-Bing J., 2013, The effects of trace elements on the lipid productivity and fatty acid composition of Nannochloropsisoculata, Journal of Renewable Energy, 2013: 1-6.

https://doi.org/10.1155/2013/671545

Zeng X.J., Chu Z.S., Yan F., and Qingru, 2009, Effects of lanthanum(III) and EDTA on the growth and competition of Microcystis aeruginosa and Scenedesmus quadricauda, Limnologica, 39(1): 86-93.

https://doi.org/10.1016/j.limno.2008.03.002

Zhang C., 2014, Essential functions of iron-requiring proteins in DNA replication, repair and cell cycle control, Protein & Cell, 5(10): 750-760.

https://doi.org/10.1007/s13238-014-0083-7

Zhang S., Liu P.H., Wang Y., Luo N., Zhang L., Yang X., 2014, Effects of iron, magnesium, and calcium on the growth and lipid accumulation of microalga Desmodesmus sp. WC08, Guangdong Agricultural Sciences, 41(4): 126-130.

Zhao Q., Ran C.G., Zhao J.W., Zhang Y.K., Xie T.H., 2020, Screening of calcifying microalgae and effect of Ca²⁺ concentration on growth and calcification, Modern Chemical Industry, 40(04): 6.

Zhou W.G., Ruan R.S., 2014, Advances and trends in microalgae carbon sequestration technologies, Science China: Chemistry, 44(1): 63-78.

https://doi.org/10.1360/032013-256