Effects of Nitrogen and Micronutrients on Spirulina platensis Physiology

Effects of Nitrogen and Micronutrients on Spirulina platensis Physiology

Nishant^1*, Shweta², Sheeba³, Purushothaman R⁴

¹PDM University, Haryana

²Starex University, Gurugram

³Manav Rachna International Institute of Research and Studies, Faridabad

⁴Annamalai University, Tamil Nadu

^*Corresponding Author

DOI: https://doi.org/10.51244/IJRSI.2025.120700090

Received: 23 June 2025; Accepted: 01 July 2025; Published: 04 August 2025

ABSTRACT

Spirulina platensis, a filamentous cyanobacterium, is extensively cultivated for its nutritional value and bioactive compounds. Environmental stressors significantly affect its growth and biochemical composition. This study examines the impact of nitrogen (NaNO₃) and micronutrient (MgSO₄, CaCl₂) stress on biomass production, specific growth rate, and chlorophyll a content in S. platensis. Cultures were grown in modified Zarrouk’s medium with fivefold increased nutrient concentrations. Growth was monitored spectrophotometrically at 565 nm (biomass), 680 nm (chlorophyll-a), and 750 nm (growth rate). Results revealed that NaNO₃ stress reduced growth and pigment accumulation, while MgSO₄ and CaCl₂ stress enhanced both. The highest biomass (0.8851 g/L) was recorded under CaCl₂ stress, and the lowest (0.2754 g/L) under NaNO₃ stress. These findings highlight the potential of micronutrient enrichment in optimizing Spirulina cultivation.

Keywords: Spirulina platensis, nitrogen stress, micronutrient stress, biomass production, chlorophyll-a

INTRODUCTION

Cyanobacteria, such as Spirulina platensis, are oxygenic photosynthetic bacteria that have oxygenated Earth for over 3 billion years (Rasmussen et al., 2008). These microorganisms are very common in freshwater and marine ecosystems, where they tend to bloom and colonize (Whitton & Potts, 2012). Arthrospira (Spirulina) platensis is a helical, filamentous, multicellular, blue-green microalga that grows in hot temperatures, strong sunlight, and alkaline conditions (Habib et al., 2008). Spirulina is highly valued for its rich nutritional content, including up to 70% protein on a dry weight basis, along with essential amino acids, fatty acids, minerals (e.g., iron, copper, zinc), vitamins (notably B12), antioxidant pigments (e.g., phycobiliproteins, carotenoids, and chlorophyll-a), and polysaccharides (Vonshak, 1997). Its commercial uses are expanding, including applications in human dietary supplements, animal feeds (terrestrial, freshwater, and marine), and pharmaceuticals. In aquaculture, Spirulina is typically used as a growth performance enhancer, pigmentation promoter, and probiotic agent in feed (Ghaeni et al., 2011; Ansarifard et al., 2018).

Microalgae such as Spirulina are heavily dependent on environmental conditions including light intensity, temperature, pH, and available nutrients for their growth and biochemical composition (Abd El-Baky et al., 2009). Here, among all, nutrient stress in terms of limitation or even excess can induce dramatic physiological changes in growth as well as pigment synthesis (Sukenik et al., 1991). Nitrogen is an essential macronutrient needed for protein and photosynthetic pigment synthesis. Micronutrients such as magnesium and calcium are essential as cofactors in the enzymatic reactions of photosynthesis (Behrenfeld & Falkowski, 1997). Furthermore, trace metals like iron (Fe), manganese (Mn), cobalt (Co), zinc (Zn), copper (Cu), and nickel (Ni) play crucial roles in a variety of metabolic processes supporting cellular function and growth (Bruland et al., 1991; Rastar et al., 2018). Specifically, iron has been found to increase cyanobacterial growth and enhance photosynthesis as well as nitrogen fixation (Rueter & Petersen, 1987).

This research seeks to determine the physiological behaviour of S. platensis under different concentrations of nitrogen (NaNO₃) and chosen micronutrients (MgSO₄ and CaCl₂). By establishing biomass yield, growth rate at specific time, and chlorophyll-a content under these stress levels, this research hopes to guide the strategies for the optimization of Spirulina large-scale cultivation in biotechnological and aquaculture practices (Bavatharny Thevarajah et al., 2022).

MATERIALS AND METHODS

The strain of S. platensis was obtained from the Centre for Conservation and Utilisation of Blue Green Algae, IARI, New Delhi

Media preparation: In the batch culture cultivation, the modified Zarrouk’s medium (Soni et al., 2019) was used with three variations of the initial nitrogen concentration and micronutrient concentration (NaNO₃, MgSO₄ & CaCl₂): 1.25g, 0.30g and 0.060g respectively represented in Table 1.

Experimental Setup: Culture conditions and growth The alga growing apparatus consists of a horizontal glass surface on the Erlenmeyer flasks had been placed. Erlenmeyer flasks of 100 ml capacity have prepared containing S. platensis (10%) with initial optical density 0.019 (Biomass concentration of 0.002 g L -1 dry weight) and 200 ml Zarrouk media (Zarrouk, 1966) at temperature 32 ˚C, pH 8.7, salinity 20 ppt with an illumination of 2500 lux light intensity, with a light/dark cycle of 12/12 h (Nhu et al., 2014). Fresh air was pumped into the solution through plastic tubes to avoid the generation of alga film layer on the wall of the flasks for 42 days, with samples collected every three days. Each stress condition was tested in triplicate.

Table. 1 Composition of modified Zarrouk Medium

Stress	Component	Normal Conc.	Stress Conc.
1st	NaNO3	0.75g	1.25g
2nd	MgSO4	0.06g	0.30g
3rd	CaCl2	0.012g	0.060g

Growth Parameter Analysis

Biomass Determination

Daily biomass concentration was assessed by measuring the optical density (OD) at 565 nm using a UV-Vis spectrophotometer. A standard curve correlating OD₅₆₅ readings with dry weight (g/L) was established by preparing serial dilutions of S. platensis cultures. This curve facilitated the estimation of biomass concentrations in experimental samples based on their OD₅₆₅ values.

Dry Weight Estimation

For dry weight determination, 15 mL of culture was filtered through pre-weighed Whatman GF/C filter papers (pore size 1.2 µm). Post-filtration, filters were rinsed twice with distilled water to remove residual salts. The filters were then dried at 80°C for 4 hours in a hot air oven and cooled in a desiccator before reweighing. The increase in filter weight corresponded to the dry biomass, expressed in g/L.

Chlorophyll-a Content Estimation

Chlorophyll-a (Chl-a) content was determined following pigment extraction using 90% acetone. A 10 mL aliquot of culture was centrifuged at 4000 rpm for 10 minutes to pellet the cells. The pellet was resuspended in 5 mL of 90% acetone and incubated in the dark at 4°C for 24 hours to facilitate pigment extraction. Post-incubation, samples were centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected. Absorbance readings were taken at 630 nm, 645 nm, and 665 nm against a 90% acetone blank. Chl-a concentration was calculated using the equation:

Chl-a (mg/L) = 11.6 × A₆₆₅ − 1.31 × A₆₄₅ − 0.14 × A₆₃₀

Specific Growth Rate (μ) Calculation

The specific growth rate (μ) was calculated during the exponential growth phase using the formula:

μ = (ln X₂ − ln X₁) / (t₂ − t₁)

Where:

X₁ and X₂ are the biomass concentrations (g/L) at times t₁ and t₂, respectively.

RESULTS

Morphological Observations

Under all treatment conditions, Spirulina platensis retained its characteristic spiral morphology. However, visual changes indicated physiological stress in certain treatments. Cultures subjected to NaNO₃ stress turned yellowish-green by day 35 and became whitish by day 42, signifying progressive stress and possible pigment degradation. In contrast, cultures treated with MgSO₄ and CaCl₂ remained green and appeared healthy throughout the experimental duration.

Biomass Concentration

Biomass accumulation was significantly affected by the type of nutrient stress in Figures 1–3. NaNO₃ stress resulted in delayed growth and a reduced final biomass concentration of 0.2754 g/L, compared to the control (0.4423 g/L) in Table 2. In contrast, MgSO₄ and CaCl₂ stress enhanced biomass production to 0.8175 g/L and 0.8851 g/L, respectively. This suggests that excess nitrogen may inhibit biomass synthesis, possibly due to metabolic imbalance, whereas elevated magnesium and calcium concentrations promote growth by supporting essential enzymatic and structural cellular functions.

Figure 1. Effect of NaNO_3​ on Biomass Production Over Time

Figure 2. Effect of MgSO₄​ on Biomass Production Over Time

Figure 3. Effect of CaCl₂​ on Biomass Production Over Time

Table 2. Average Biomass under control and stress conditions

Treatment	Control (g/l)	Stress (g/l)
NaNO₃	0.4423	0.2754
MgSO₄	0.4934	0.8175
CaCl₂	0.4563	0.8851

Chlorophyll-a Concentration

Chlorophyll-a levels varied notably under different stress conditions (Figures 4–6). Under NaNO₃ stress, the pigment concentration decreased slightly to 1.0557 mg/L, while MgSO₄ and CaCl₂ treatments elevated chlorophyll-a to 1.1212 mg/L and 1.1203 mg/L, respectively (Table 3). Compared to control values (~1.077 mg/L), these findings imply that magnesium and calcium supplementation enhances pigment biosynthesis, possibly through their role as cofactors in chlorophyll and protein synthesis pathways. In contrast, nitrogen excess may suppress pigment formation due to cellular toxicity or imbalance in the nitrogen assimilation process.

Figure 4: Effect of NaNO₃ Stress on Chlorophyll-a Concentration

Figure 5: Effect of MgSO₄ Stress on Chlorophyll-a Concentration

Figure 6: Effect of NaNO₃ Stress on Chlorophyll-a Concentration

Table 3. Average Chlorophyll-a Concentration

Treatment	Control (mg/L)	Stress (mg/L)
NaNO₃	1.0776	1.0557
MgSO₄	1.0781	1.1212
CaCl₂	1.0778	1.1203

Specific Growth Rate

Specific growth rate (µ) analysis further emphasized the differential effects of nutrient stress (Figures 7–9). The lowest growth rate was recorded under NaNO₃ stress (µ = 0.0055 day⁻¹), accompanied by an extended generation time of 107 hours. In contrast, MgSO₄ significantly improved growth (µ = 0.0228 day⁻¹; generation time = 44 hours), followed by CaCl₂ (µ = 0.0136 day⁻¹; generation time = 282 hours) (Table 4). These results confirm that magnesium and calcium not only enhance biomass but also support faster population doubling, possibly due to their involvement in photosynthesis and structural stability.

Figure 7. Effect of NaNO₃ Stress on Specific Growth Rate

Figure 8. Effect of MgSO₄ Stress on Specific Growth Rate

Figure 9. Effect of CaCl₂ Stress on Specific Growth Rate

Table 5. Growth Parameters under Stress Conditions

Parameter	NaNO₃	MgSO₄	CaCl₂
Avg Biomass (g/L)	0.2754	0.8175	0.8851
Specific Growth Rate µ	0.0055	0.0228	0.0136
Generation Time (h⁻¹)	107	44	282

Statistical Analysis

Standard deviation analysis (Figure 10) for both biomass and chlorophyll-a measurements under control and stress conditions revealed low variability across all treatments, reflecting high reproducibility. Among the stress treatments, MgSO₄ and CaCl₂ exhibited lower standard deviation values compared to NaNO₃, indicating more stable growth and pigment profiles under these conditions.

Figure 10. Standard Deviation of Biomass and Chlorophyll-a in Spirulina platensis Under Control and NaNO₃ Stress Conditions

DISCUSSION

Nitrogen plays a central role in the metabolism of Spirulina platensis, directly influencing biomass accumulation, pigment synthesis, and enzymatic activity. While earlier studies suggested that increased nitrogen enhances protein, phycocyanin, and lipid synthesis but represses carotenoid production due to a metabolic shift (Kand & Nagarajan, 2013), our study observed that an excess of NaNO₃ actually suppressed biomass, chlorophyll-a levels, and growth rate. This may be attributed to nitrogen toxicity, which causes metabolic imbalance, oxidative stress, and potential ammonia accumulation, negatively affecting cellular functions (Yadav et al., 2021). On the other hand, supplementation with micronutrients such as MgSO₄ and CaCl₂ significantly improved growth parameters, indicating their beneficial roles. Magnesium, as the central atom of chlorophyll and a cofactor for RuBisCO and ATPase, enhances photosynthetic efficiency and energy metabolism (Hu et al., 2020), while calcium stabilizes membrane integrity, modulates ion channels, and acts as a secondary messenger in stress signaling (Singh et al., 2022). These ions not only support pigment biosynthesis and protein function but also help mitigate oxidative damage under stressful conditions. Our findings align with recent studies that demonstrate how optimizing nutrient stoichiometry (particularly N:Mg:Ca ratios) enhances microalgal resilience and metabolic output (Li et al., 2023). Therefore, careful modulation of nutrient levels—rather than excess—offers a practical route to maximize S. platensis productivity for biotechnology and aquaculture applications.

Nitrogen starvation usually results in physiological responses including reduced phycocyanin production and enhanced carotenoid and exopolysaccharide production, possibly as defense mechanisms against stress (Solovchenko et al., 2008). Micronutrients like iron, magnesium, calcium, and zinc are responsible for regulating these stress reactions through the provision of enzymatic functions and biosynthesis of pigments. For instance, iron is required for chlorophyll synthesis and redox processes in photosynthesis; its deprivation can lead to serious growth inhibition (Panyakampol et al., 2016).

In our work, elevated levels of NaNO₃ in Zarrouk’s medium resulted in repressed growth with a maximal biomass concentration as low as 0.2754 g/L. In both MgSO₄ and CaCl₂ treatments, significantly enhanced biomass concentrations were obtained, viz., 0.8175 g/L and 0.8851 g/L respectively. These findings attest to the degradative influences of excessive nitrogen and the enhancing growth advantage from magnesium and calcium supplementation. Interestingly, there were no morphological alterations among Spirulina filaments throughout the treatments, showing that physiological rather than structural parameters were affected mainly by stress conditions.

Chlorophyll-a content followed the same trends as biomass data. In the NaNO₃ stress condition, chlorophyll-a decreased slightly to 1.0557 mg/L, while MgSO₄ and CaCl₂ treatments increased pigment levels to 1.1212 mg/L and 1.1203 mg/L, respectively—about double the initial values.

Specific growth rate (µ) analysis also corroborated these findings. NaNO₃-stressed cultures had the lowest growth rate (0.0055 day⁻¹), whereas MgSO₄-stressed cultures had the highest (0.0228 day⁻¹). CaCl₂ also stimulated growth (0.0136 day⁻¹), albeit less so. Generation time s(doubling time) was shortest in MgSO₄ stress (44 hours) and longest in CaCl₂ stress (282 hours), indicating variations in metabolic efficiency.

In general, the data show that controlled enrichment with micronutrients, as opposed to nitrogen increase, is a superior method for maximizing Spirulina growth and pigment yield. These results indicate that accurate management of nutrients can have a major impact on increasing the productivity of Spirulina culture systems under normal conditions (33 °C, 1900 cd·sr/m² light intensity, 18:6 light/dark cycle, and pH 8.7).

Statistically, the variability between treatments was moderate, confirming the stability of the experimental design. Yet, future research needs to try to minimize control condition variability further and investigate molecular mechanisms behind stress responses in Spirulina in order to tailor cultivation strategies even more precisely.

Limitations and future study: While this study provides valuable insights into the individual effects of nitrogen and micronutrient stress, it is limited by its laboratory-scale design and single-stressor focus. Natural environments present more complex conditions involving multifactorial stresses such as light fluctuations, temperature shifts, and nutrient interactions. Future research should explore combined stressors, particularly interactions between macronutrients and micronutrients, and validate findings in semi-field or outdoor cultivation systems. Molecular-level investigations into stress-responsive gene expression could also yield a deeper understanding of S. platensis adaptability.

CONCLUSION

This work proves that micronutrient supplementation MgSO₄ and CaCl₂ increases S. platensis biomass and chlorophyll-a content, whereas a surplus of nitrogen (NaNO₃) inhibits growth. This information is important for maximizing large-scale cultivation practices in aquaculture and biotechnology. Spirulina platensis stress physiology is highly dependent on the availability of nitrogen and micronutrient levels with important consequences for growth, biochemical structure, and pigment accumulation. Nitrogen stress regulates protein, phycocyanin, and carotenoid synthesis, whereas micronutrient stress affects enzyme function, fatty acid composition, and carbohydrate production. Optimizing growth conditions requires knowledge of these responses to augment the production of valuable compounds like phycocyanin and carotenoids. Future research must investigate combined effects of multiple micronutrients and light regimens to maximize Spirulina productivity.

Summary table:

Treatment	Avg. Biomass (g/L)	Chlorophyll-a (mg/L)	Growth Rate µ (day⁻¹)	Generation Time (h)
Control	~0.45	~1.08	—	—
NaNO₃	0.2754	1.0557	0.0055	107
MgSO₄	0.8175	1.1212	0.0228	44
CaCl₂	0.8851	1.1203	0.0136	282

Author Contributions: The first draft of the manuscript was written by First author and all authors commented on previous versions. All authors read and approved the final manuscript.

ACKNOWLEDGEMENT

We acknowledge the authorities of all universities of authors to all the support to carry out the work.

Declaration of Interest statement:

Funding: Not applicable

Competing Interest: The authors have no relevant financial or non-financial interests to disclose

Ethics approval: Not applicable

Constant to Publish: Not applicable

Conflict of Interest Statement: None declared

Data availability statement: Data available on request

REFERENCES

1. Abd El-Baky, H. H., El Baz, F. K., & El-Baroty, G. S. (2009). Enhancement of antioxidant production in Spirulina platensis under oxidative stress. Acta physiologiae plantarum, 31, 623-631.
2. Ansarifard, F., Rajabi Islami, H., Shamsaie Mehrjan, M., & Soltani, M. (2018). Effects of Arthrospira platensis on growth, skin color and digestive enzymes of Koi, Cyprinus carpio. Iranian Journal of Fisheries Sciences, 17(2), 381-393.
3. Baldia, S. F. (1991). Effects of physico-chemical factors and nutrients on the growth of Spirulina platensis isolated from Lake Kojima, Japan. 日本水産学会誌, 57(3), 481-490.
4. Bruland, K. W., Donat, J. R., & Hutchins, D. A. (1991). Interactive influences of bioactive trace metals on biological production in oceanic waters. Limnology and oceanography, 36(8), 1555-1577.
5. Chojnacka, K., & Noworyta, A. (2004). Evaluation of Spirulina sp. growth in photoautotrophic, heterotrophic and mixotrophic cultures. Enzyme and microbial technology, 34(5), 461-465.
6. Esen, M., & Ozturk Urek, R. (2015). Ammonium nitrate and iron nutrition effects on some nitrogen assimilation enzymes and metabolites in Spirulina platensis. Biotechnology and Applied Biochemistry, 62(2), 275-286.
7. Ghaeni, M., Matinfar, A., Soltani, M., Rabbani, M., & Vosoughi, A. (2011). Comparative effects of pure spirulina powder and other diets on larval growth and survival of green tiger shrimp, Peneaus semisulcatus.
8. Habib, M. A. B., Parvin, M., Huntington, T. C., & Hasan, M. R. (2008). A review on culture, production and use of Spirulina as food for humans and feeds for domestic animals.
9. Hu, X., Wu, M., Hong, D., Xue, X., Zhao, M., Gao, Y., … & Huo, S. Unraveling the Dual Pathways of Intracellular Nitrite Accumulation in Microalgae Under Free Nitrous Acid (Fna) and Nitrite Salt (No2-) Stress. Available at SSRN 5271593.
10. Li, Q., et al. (2023). Nutrient stoichiometry regulates photosynthetic pigment and antioxidant capacity in Spirulina. Bioresource Technology Reports, 22, 101331.
11. Moraes, I. D. O., Arruda, R. D. O. M., Maresca, N. R., Antunes, A. D. O., & Moraes, R. D. O. (2013). Spirulina platensis: process optimization to obtain biomass. Food Science and Technology, 33, 179-183.
12. Munawaroh, H. S. H., Fathur, R. M., Gumilar, G., Aisyah, S., Yuliani, G., Mudzakir, A., & Wulandari, A. P. (2019, November). Characterization and physicochemical properties of chlorophyll extract from Spirulina sp. In Journal of Physics: Conference Series(Vol. 1280, No. 2, p. 022013). IOP Publishing.
13. Nhu, T. N. H., & Hiep, N. H. (2014). The effect of pH, dark-light cycle and light colour on the chlorophyll and carotenoid production of Spirulina sp. KKU Res. J, 19(3), 190-197.
14. Olguı́n, E. J., Galicia, S., Angulo-Guerrero, O., & Hernández, E. (2001). The effect of low light flux and nitrogen deficiency on the chemical composition of Spirulina sp.(Arthrospira) grown on digested pig waste. Bioresource technology, 77(1), 19-24.
15. Panyakampol, J., Cheevadhanarak, S., Senachak, J., Dulsawat, S., Siangdung, W., Tanticharoen, M., & Paithoonrangsarid, K. (2016). Different effects of the combined stress of nitrogen depletion and high temperature than an individual stress on the synthesis of biochemical compounds in Arthrospira platensis C1 (PCC 9438). Journal of Applied Phycology, 28, 2177-2186.
16. Rasmussen, B., Fletcher, I. R., Brocks, J. J., & Kilburn, M. R. (2008). Reassessing the first appearance of eukaryotes and cyanobacteria. Nature, 455(7216), 1101-1104.
17. Rastar, M., Hosseini Shekarabi, S. P., Shamsaie Mehrgan, M., & Sabzi, S. (2018). Effects of iron and zinc concentrations on growth performance and biochemical composition of Haematococcus pluvialis: A comparison between nanoparticles and their corresponding metals bulks. Journal of Algal Biomass Utilization, 9(2), 59-67.
18. Rueter, J. G., & Petersen, R. R. (1987). Micronutrient effects on cyanobacterial growth and physiology. New Zealand Journal of Marine and Freshwater Research, 21(3), 435-445.
19. Singh, R., et al. (2022). Calcium-mediated signaling under abiotic stress in cyanobacteria: Recent insights. Plant Physiology and Biochemistry, 178, 19–29.
20. Solovchenko, A. E., Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., & Merzlyak, M. N. (2008). Effects of light and nitrogen starvation on the content and composition of carotenoids of the green microalga Parietochloris incisa. Russian Journal of Plant Physiology, 55, 455-462.
21. Soni, R. A., Sudhakar, K., & Rana, R. S. (2019). Comparative study on the growth performance of Spirulina platensis on modifying culture media. Energy Reports, 5, 327-336.
22. Sujatha Kand, S. K., & Nagarajan, P. (2013). Effect of different nitrogen concentrations on the biomass and biochemical constituents of Spirulina platensis [Geitler].
23. Sukenik, A., & Wahnon, R. (1991). Biochemical quality of marine unicellular algae with special emphasis on lipid composition. I. Isochrysis galbana. Aquaculture, 97(1), 61-72.
24. Thevarajah, B., Nishshanka, G. K. S. H., Premaratne, M., Nimarshana, P. H. V., Nagarajan, D., Chang, J. S., & Ariyadasa, T. U. (2022). Large-scale production of Spirulina-based proteins and c-phycocyanin: A biorefinery approach. Biochemical Engineering Journal, 185, 108541.
25. Vonshak, A. (Ed.). (1997). Spirulina platensis arthrospira: physiology, cell-biology and biotechnology. CRC press.
26. Whitton, B. A., & Potts, M. (2012). Introduction to the cyanobacteria. In Ecology of cyanobacteria II: their diversity in space and time(pp. 1-13). Dordrecht: Springer Netherlands.
27. Yadav, G., et al. (2021). Nitrogen metabolism and regulation in cyanobacteria under environmental stress. Frontiers in Microbiology, 12, 645925.