The microalgae industry is continuously seeking sustainable, non-mineral-based nutritional sources to lower production costs while enhancing overall sustainability. Multiple waste streams have been evaluated and are used currently worldwide in large-scale microalgae farms. Different waste streams showed high potential for microalgae cultivation, which include food waste (Kumar et al., 2022), municipal wastewater (Han et al., 2024), anaerobic digestate (Bauer et al., 2021), and waste glycerol (Xu et al., 2019). In addition, microalgae showed high growth capability on high salinity water, including brine, i.e., the discharge of desalination plants (Al Bazedi et al., 2023). In 2017, there were about 19,372 desalination plants globally, a collective capability of producing around 99.8 million m³ of freshwater per day (WaterWorld, 2017). Per each liter of produced fresh water, there are about 1.5 L of highly concentrated brine produced (Jones et al., 2019). However, a significant environmental concern arises from the brine generated as a byproduct of desalination processes. Brine disposal methods commonly include sewer discharge, evaporation ponds, surface water discharge, deep-well injection, and land application (Mickley, 2018). Several studies have evaluated the adverse environmental effects of brine disposal on marine ecosystems, groundwater, and soil quality. Potential environmental damage may include pH fluctuations, eutrophication, and increased levels of heavy metals in marine environments and plant/animal mortalities (Panagopoulos et al., 2019).

In order to meet the carbon footprint derivatives of the European Union, many European countries aim to produce green energy from hydrogen. Sustainability will largely depend on the use of seawater, which is available in unlimited quantities and does not compete with water supply for human consumption. However, using seawater for hydrogen production requires prior desalination. This amount will comprise a problem since there is no concept for disposal of the produced brine. This may become the bottleneck for the entire energy transformation plans in countries counting on green hydrogen energy. Thus, finding new alternatives to remove and valorize brine eco-friendly and cost-effectively are of great importance. A recent approach is to convert brine into valuable biomass through microalgae cultivation in brine-based growth media. Several species such as Chlorella vulgaris, Scendesmus quadricauda, A. platensis, Nannochloropsis oculata, and Dunaliella tertiolecta have successfully been grown on brine-based media (Al Bazedi et al., 2023). Moreover, some microalgae, when cultivated under salinity stress, enhance the production of valuable compounds such as pigments, lipids, carbohydrates, and other bioproducts. This opens possibilities not just to reduce the volume of rejected brine but also to reduce production costs associated with prices for micronutrient media (Osman et al., 2023).

The microalgae market in European countries can not currently compete with the low-cost systems used e.g., in Asia, which limits market applications. Cyanobacteria like A. platensis have a high tolerance to elevated salinity. For instance, some A. platensis strains showed slightly reduced growth at 600 mM NaCl (3.5% salinity, similar to seawater) (Liu et al., 2016). Experiments showed that content of 5%–15% seawater in the culture medium only causes a decrease of biomass yield by 15%–20% as compared with Zarrouk’s culture medium (Tambiev et al., 2011). A. platensis has a unique quality to detoxify (neutralize) or to chelate toxic minerals and heavy metals (Okamura and Aoyama, 1994), and is also capable of removing minerals from inland saline waters (Sandeep et al., 2013).

Another cost driver in microalgae production is the source of macronutrients, i.e., nitrogen, phosphorus, and potassium. In order to reduce such costs, sustainable resources have successfully been tested in recent years. It was shown that replacing P with struvite-P enhances c-phycocyanin (C-PC) production in A. platensis, without compromising growth and biomass yield (Beyer et al., 2023). Struvite is the crystalized form of the mineral magnesium-ammonium-phosphate (MAP) with the formula NH₄MgPO₄ 6H₂O. Several waste streams can be exploited for the production of sustainable struvite, e.g., urban wastewater, industrial wastewater, manure, or livestock slurries (Huygens et al., 2019). As struvite is already included as a commercial fertilizer in the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) EC No. 1907/2006 and in the 2023 amendment to the EU eco-regulation/inclusion, the use of it in culture media is of special interest for the creation of new formulated sustainable media (European Commission, 2023).

Growth media combining both brine and struvite as micro- and macronutrient replacements have, to the best of authors’ knowledge, never been reported. If successful, such a cost-effective fully sustainable resource combination would open the opportunity for microalgae producers to enter new markets. Therefore, this study aims to test whether media combined of brine and struvite (recovered from Agri industries) is a suitable application for A. platensis production, with a special focus on C-PC production. It was cultivated on several media combinations, i.e., with brine (as a supplement to NaCl, micronutrients, and freshwater), and struvite (recovered from cow urine to replace mainly mineral P). The impacts on microalgal growth and C-PC yield were compared to the control groups using the standard SAG growth medium.

The present study is the first report proofing that A. platensis can be grown on local waste streams fully replacing mineral-based synthetic growth medium with struvite and brine. This would help the agri-industry sector to valorize and reduce waste streams from animal production, i.e., cow urine. In addition, it would provide the growing biohydrogen industry with a biological approach to valorize and reduce brine in the coming decades. On the other hand, the new suggested media will provide the microalgae industry with a near-zero cost growth medium at simultaneous high sustainability. This implies that brine can replace micronutrients coming from commercial micronutrient pre-mixes. It also implies that production can even be enhanced when commercial media and brine are used together.

Comparing the obtained maximum specific growth rate of A. platensis to values in literature, the present values using YLT in SAG showed 0.1808 days⁻¹, which is higher than the stated values of the maximum specific growth rate of about 0.144 days⁻¹ (Islam et al., 2003; Usharani et al., 2012). Results from the modified culture media BS I and BS III under YLT showed a significant increase in growth compared to the control group (SAG). Interestingly, using struvite and diluted brine as main nutrient sources in BS III resulted in a high maximum growth rate of 0.22 days⁻¹, showing that both positively influence the growth of A. platensis. Interestingly, BS III, despite its minimal composition, performs nearly similar to BS I under yellow-white light. This indicates that A. platensis can effectively utilize the nutrients provided by struvite and brine, potentially reducing the need for additional nutrient supplementation. The slightly lower performance of BS II compared to BS I and BS III under yellow-white light might suggest some benefit from either the presence of micronutrients (as in BS I) or their complete absence (as in BS III), though this difference appears minor. The effectiveness of BS III can be attributed to the complementary nutrient profiles of struvite and brine. Struvite provides essential macronutrients (N, P, and Mg), while the brine serves as an excellent source of micronutrients, including Zn, Mn, B, Mo, and Fe, which are crucial for various metabolic processes in A. platensis. This synergistic nutrient provision likely contributes to the enhanced growth observed. Several studies demonstrated that Brine discharges from reverse osmosis plants include trace metals, such as iron (Fe), nickel (Ni), chromium (Cr), and molybdenum (Mo) (Omerspahic et al., 2022). The composition of the brine used in this study is presented in Table 1. When compared to brine compositions reported in other studies (Ahmed et al., 2001; Rivero-Falcón et al., 2023), brine used in the present study shows similarities in potassium and sodium levels, but differences in other elements. Notably, our brine contains lower levels of iron, manganese, and zinc, but detectable amounts of Mo and vanadium, which are not reported in the reference sources. Nitrogen content also varies significantly among the sources. It's important to note that brine composition can vary significantly depending on the location of the desalination plant, the seawater composition, and the technique used for desalination. This variability is evident in the diverse compositions reported in literature (as seen in Table 1). Consequently, these results, while promising, cannot be universally applied to microalgae media development based on brine without considering these compositional variations.

Recent studies have explored the use of struvite as an optimized nutrient source for microalgae cultivation. Struvite has been shown to support biomass productivity comparable to or higher than conventional media for various microalgae species (Chaoyu et al., 2017; Tang et al., 2023). It provides efficient nutrient utilization and can satisfy trace metal requirements (Davis et al., 2015). Moreover, the controlled nutrient release properties of struvite make it a valuable buffering nutrient source, with the potential to enhance the growth of microalgae (Tang et al., 2023). Experiments were carried out to assess the use of struvite as a primary nutrient source for cultivating two industrially relevant microalgae species: Nannochloropsis salina and Phaeodactylum tricornutum. These trials were conducted in both laboratory settings and outdoor pilot-scale raceways, spanning various seasonal conditions. The results demonstrated that media formulations containing either raw or refined struvite supported biomass production rates that were comparable to, or exceeded, those achieved with conventional growth media. In outdoor co-culture conditions, the peak biomass yield reached approximately 20 ± 4 g of ash-free dry weight per square meter per day. It demonstrates that these waste-derived nutrient sources can effectively support A. platensis growth, potentially as well as or better than conventional synthetic media. This finding supports the main thesis of using waste streams for sustainable microalgae cultivation (Davis et al., 2015). However, growth rates under BLT showed lower values compared to values reported in the literature (Islam et al., 2003; Usharani et al., 2012). This shows that blue light is less beneficial for the growth of A. platensis than yellow light. This can be attributed to the differences in the action spectra of photosynthesis (Jorgensen et al., 1987; Subramaniam et al., 1999). Although both light treatments had similar irradiances in the PAR region, the photosynthetic efficiency under yellow light is generally higher due to the action spectra of photosynthesis (Blanken et al., 2013). This difference can be attributed to several factors, a) Absorption efficiency where yellow light (570–590 nm) is more efficiently absorbed by chlorophyll a and phycobilins, the primary photosynthetic pigments in A. platensis (Markou et al., 2016). b) Photosystem balance where yellow light may provide a more balanced excitation of both photosystem I and II, potentially leading to more efficient electron transport and ATP and NADPH production, the essential molecules for carbon fixation and biomass accumulation, compared to blue light (Mohsenpour and Willoughby, 2013). This difference in photosynthetic response likely explains the increased biomass productivity observed under yellow light in the present study. Research on A. platensis cultivation under different light conditions reveals varying effects on biomass and pigment production. Red light generally promotes the highest biomass concentration and pigment production per culture volume (Tayebati et al., 2021). However, BLT results in the highest pigment content per dry weight, despite lower overall biomass (Tayebati et al., 2021). YLT enhances phycocyanin and allophycocyanin production in mixotrophic cultures (Yun et al., 2021). White light yields the highest biomass in mixotrophic conditions but lower pigment concentrations (Rizzo et al., 2015; Yun et al., 2021). Light quality significantly influences energy transfer processes within photosystems (Haury and Bogorad, 1977). Yellow light, in particular, enhances the energy transfer efficiency in phycobilisomes, which are connected to Photosystem II, as observed in both cyanobacteria and red algae (Akimoto et al., 2012; Biswas et al., 2024). This efficient energy transfer under yellow light is closely tied to the action spectra for photosynthesis, contributing to the overall photosynthetic efficiency and potentially explaining the higher biomass productivity observed in the present study.

While these results clearly demonstrate enhanced biomass and PC productivity under YLT compared to BLT, the underlying mechanisms require further investigation. The observed effects could be due to increased photosynthetic efficiency, photomorphogenic responses, or a combination of both. Photomorphogenesis, which involves gene induction in response to specific light qualities, can significantly influence the accumulation of bioactive compounds like PC. Previous studies have shown that blue or red light can induce photomorphogenic responses that enhance PC production (Pagels et al., 2020a; Pagels et al., 2020b). In order to distinguish between these processes, future experiments could adopt a photo biological strategy where photosynthesis is saturated under yellow light, followed by the application of low irradiances of blue or red light to evaluate their photomorphogenic effects. This approach would clarify the extent to which the observed increases in biomass and PC production under YLT are attributable to photosynthetic activity versus photo morphogenesis.

Overall, the growth of A. platensis in media enriched with brine (in addition to SAG micronutrients, BSI) was significantly higher than that in the SAG medium. However, the growth in media containing solely brine (BSIII, without micronutrients) was similar to the growth in the SAG medium. Previous reports showed that the effect of salinity on microalgae including Arthrospira varies. For example, optimal salinity levels for maximum biomass productivities of Oocystis pusilla were observed at around 3,000 ppm Total Dissolved Solids (TDS), resulting in significant increases in biomass yield compared to standard media. However, salinity levels beyond this optimum (e.g., 3,500 ppm TDS) led to marked reductions in productivity (Osman et al., 2023). Species-specific responses have been noted, with A. maxima showing continuous decreases in growth with increasing seawater levels, where final dry biomass decreased by 15% below the control (Tambiev et al., 2011). In addition, the growth of A. platensis completely stopped at seawater concentrations ranging between 40% and 60%. Liu et al. (2016) reported similar growth in A. platensis cultivated at low salinity (200 and 400 mM NaCl) to those obtained in the control Zarrouk medium, with slightly lower growth (−6%) at a salinity of 600 mM. A similar effect, decreasing growth and biomass yield, with increasing salinity, was reported in other studies (Polat et al., 2020; Tejada-Ruiz et al., 2020; Ghalhari et al., 2022).

The present salinity values that supported the highest growth in A. platensis were higher than those reported to inhibit the growth. One factor that might contribute to enhanced growth in the present study is the enrichment of brine with microalgal nutrients. Previous studies reported enhanced microalgal growth in brine. For instance, the growth of C. vulgaris enhanced when grown in 25% dilution of brine mixed with bold basal media (BBM), while brine dilutions of 30%–35% resulted in growth inhibition (Gilani et al., 2024). However, Tambiev et al. (2011) utilized sea salt sourced from Black Sea with Zarrouk’s medium at various volumetric ratios. Results confirmed the inhibitory effects of natural, pre-filtered, and autoclaved sea salt. Notably, the strain utilized in the present study (UTEX 2340) was not specifically previously evaluated for its high salt tolerance. These findings suggest that the strain exhibits a noteworthy tolerance to high salinity levels, which would play a crucial role in future applications.

Apart from the impact of struvite-brine-based media (SBBM) on growth, the present study aimed to evaluate if the SBBM potentially compromises C-PC enhancing effect of struvite recorded in the recent study (Beyer et al., 2023). Results confirmed that SBBM has no negative impact on C-PC yield. This may be related to the passive transfer of ammonia into the cell which may be independent of other minerals affecting active ion transfer across the cell membrane. As previously suggested (Beyer et al., 2023), higher C-PC is correlated to a nitrogen-sparing effect associated with ammonia fraction in struvite. The present study also demonstrated that SBBM enhanced C-PC production, especially during the exponential growth phase. This has an important industrial value since the harvesting time of biomass and C-PC will be towards the end of this phase. C-PC measurements were done mainly to confirm that there is no negative effect on C-PC associated with brine in the media and this was confirmed. Recent studies on A. platensis cultivation have revealed strategies to enhance both biomass and C-phycocyanin (C-PC) production. Light intensity significantly affects growth and product formation, with lower intensities favoring C-PC production and higher intensities promoting extracellular polymeric substances (Dejsungkranont et al., 2017). Optimal light intensities for cell growth (282 μmol m⁻² s⁻¹) and phycocyanin synthesis (137 μmol m⁻² s⁻¹) differ, with light attenuation impacting intracellular phycocyanin content (del Rio-Chanona et al., 2015). Research on light quality and intensity effects on microalgal cultivation has shown promising results for enhancing biomass and phycocyanin production. White light in mixotrophic conditions yielded the highest biomass concentration for A. platensis (Chainapong et al., 2012). However, yellow and red lights in mixotrophic cultures produced the highest phycocyanin content (Chainapong et al., 2012). White light generally results in higher biomass and phycocyanin production compared to green and blue light (Jung et al., 2022). Moreover, the overall yield was not improved under BLT due to the lower growth rate of A. platensis. These results highlight the complex interplay between nutrient availability, light conditions, and metabolite production in A. platensis.

The aforementioned findings have significant implications for sustainable A. platensis cultivation, demonstrating that simplified, potentially more cost-effective and environmentally friendly media based on brine and struvite can support and even enhance C-PC production, particularly when combined with appropriate light conditions. The success of BS III, in particular, highlights its potential for developing highly efficient minimal growth medium for industrial-scale production of this valuable cyanophyte. The data clearly shows that yellow-white light consistently leads to higher biomass and C-PC productivity across all media types compared to the blue-white light. Under yellow-white light, the brine-struvite-based media (BS I, II, and III) outperformed the standard SAG medium, with BS I showing the highest C-PC productivity, followed closely by BS III. In contrast, under blue-white light, C-PC productivity was generally lower and more consistent across media types, with BS III showing a slight advantage. The standard SAG medium consistently yielded the lowest C-PC productivity under both light conditions. These results suggest that using brine-struvite-based media, particularly BS I or BS III, in combination with yellow-white light, could significantly enhance C-PC production in A. platensis cultures compared to the standard media and lighting conditions.

The present study marks a significant milestone by demonstrating that A. platensis can be cultivated using local waste streams, i.e., combined brine and struvite, to effectively substitute mineral-based synthetic growth mediums. Results not only offer a solution for valorizing and reducing waste streams from the agricultural industry but also present an innovative approach for the emerging hydrogen energy sector to address brine disposal challenges in the coming decades. Moreover, the novel media tested in this study provides the microalgal industry with a cost-effective and highly sustainable alternative. The incorporation of brine and struvite as nutrient sources resulted in enhanced growth rates, showing the potential synergistic effects between natural and synthetic components. Despite the variability in brine composition, the observed growth enhancements confirmed the adaptability of the cultivated A. platensis to diverse environmental conditions. Furthermore, the study confirmed that the use of SBBM does not influence the production of C-PC as a valuable pigment with many industrial applications, suggesting the need of further biotechnological exploitation. These findings not only expand the current understanding of microalgae cultivation, but also highlight the potential for integrating waste valorization and sustainable resource utilization in bioprocessing industries. The broader implications of this research are far-reaching, potentially revolutionizing multiple sectors including circular economy practices, sustainable agriculture, clean energy transition, food and nutrition security, and biotechnology. Future research directions could include scaling-up the cultivation process, investigating other microalgal species, exploring geographical variations, developing automated optimization systems, and conducting comprehensive life cycle analyses. By addressing these areas, future studies can build upon this foundational work, further advancing the fields of sustainable biotechnology and circular economy practices.