Recirculation of Saline Concentrate in Spirulina Cultivation: A Promising Strategy for High Production of Biomass and Biomolecules in Semiarid Regions

Abstract

Desalination concentrate (DC) is a residue from desalination plants in semiarid regions, and it is promising for microalgae cultures. Its effects have been evaluated in a previsions study involving Spirulina sp. LEB 18 cultures grown in a medium containing 100% DC, supplemented with 25% Zarrouk nutrients, and without reuse. In the present study, Spirulina sp. LEB 18 was cultivated in the recycled saline concentrate medium from the previous experiment over three consecutive cycles using raceways. The recycled DC achieved a maximum biomass production of 3.77 g·L⁻¹, surpassing the control production (3.45 g·L⁻¹), and demonstrated high resistance to elevated environmental temperatures, reaching 42.80 °C. Protein levels (≤37%) containing all essential amino acids (~40% of total proteins) and fatty acids with relevant impacts on health were reached. Using up to three DC cycles for Spirulina sp. cultivation can contribute to the valorization of this residue and add viability to semiarid regions for biomass production with adequate nutritional composition for supplementation of humans and animals.

1. Introduction

The gradual depletion of fresh water worldwide is attributed to the combined effects of social inequality, inadequate resource management, limited water availability, and unsustainable practices [1,2]. Semiarid regions are severely affected by depletion because of their climatic and geographical conditions, including frequent droughts. However, many of these regions possess underground reserves of brackish water from the hydrogeology of crystalline rocks and fissure aquifers [3]. To enable the use of brackish waters, desalination can be carried out, but it promotes the generation of waste known as desalination concentrate (DC).

DC has a high total salinity (>0.5 g·L⁻¹) compared with fresh water, making it unsuitable for human consumption and application to conventional agricultural systems. Improper disposal of this effluent can lead to detrimental environmental effects, such as soil and aquatic system salinization [3]. Nevertheless, DC commonly contains inorganic minerals (e.g., Cl⁻, Na⁺, and Ca²⁺), other nutrients, such as nitrogen and phosphorus, and trace elements (e.g., Si, K⁺, Mg⁺², and Fe⁺³), which contribute to the process such as the growth and metabolism of microalgae [4]. Microalgae and cyanobacteria are microorganisms that produce high-value biomass rich in lipids, proteins, carbohydrates, and pigments [5].

Among the species of microalgae, Spirulina sp. is widely used for nutritional supplementation of humans and animals, and it has a high capacity for bioremediation and desalination [6,7,8,9]. Spirulina biomass is rich in proteins (>60%) and bioactive functional constituents such as polyunsaturated fatty acids, essential amino acids, enzymes, vitamins (especially B12), minerals, and pigments (e.g., β-carotene, chlorophyll a, and phycocyanin) [9]. Mata et al. [10] demonstrated the cultivation of Spirulina sp. using DC from desalinated brackish groundwater from an artesian well in a semiarid region of Northeast Brazil, achieving high concentrations of lipids (12.79%) and carbohydrates (52.29%) in the biomass as a response to saline stress.

Further studies must be conducted on the cultivation of microalgae using reused DC (RDC) over multiple cycles to determine the viability of biomass growth and composition of biomolecules across successive cycles. Although the reuse of DC in microalgal cultivation is not a common practice, the cultivation of species such as Chlorella vulgaris [11] and Nannochloropsis gaditana [12] has been demonstrated, with notable changes in the lipid, pigment, carbohydrate, and protein contents in the microalgal biomass.

There is no available study on the reuse of DC from microalgal cultures under reduced nutritional demand in pilot-scale or outdoor systems for the cultivation of Spirulina. Therefore, we presented a novel alternative method for the outdoor cultivation of Spirulina sp. LEB 18 using RDC as the culture medium. The cultivation cycles involve DC recirculation while assessing biomass and biomolecule production.

2. Materials and Methods

2.1. Microalgae and, Water Collection

The microalgae utilized in this study was Spirulina sp. LEB 18, sourced from the Culture Collection of the Biochemical Engineering Laboratory (LEB) at the Federal University of Rio Grande, Rio Grande, Brazil. The inoculum was maintained in Zarrouk culture medium [13] at 30 °C, using constant aeration at a flow of 45 Lh⁻¹ with the air previously filtered in glass wool to remove contaminants. A photoperiod of 12 h of light and 12 h of darkness was applied, with a light intensity of 44.8 μmol m⁻²s⁻¹ using fluorescent lamps without nutrient supplementation. Water was added to compensate for evaporation losses. The culture medium was the RDC considered by Mata et al. [10], with the DC collected in the village of Mandassaia II, the city of Riachão do Jacuípe (11°54′25.8′′ S, 39°23′38.8′′ W), Bahia, Brazil.

2.2. Experimental Conditions

In [10], the cultivation of Spirulina sp. LEB 18 was carried out using 100% DC supplemented with 25% concentrations of carbon sources (NaHCO₃), nitrogen (NaNO₃), (K₂HPO₄), potassium (K₂SO₄), sodium (NaCl), magnesium (MgSO₄·7H₂O), calcium (CaCl₂), iron (FeSO₄·7H₂O), and ethylenediaminetetraacetic acid (EDTA) present in the Zarrouk culture medium [13]. After cultivation, the biomass was separated from the cultivation medium for biochemical composition analysis. The RDC medium was then analyzed for the following contaminants: lead (Pb) < 0.10 mg·L⁻¹; cadmium (Cd) < 0.005 mg·L⁻¹; nickel (Ni) < 0.02 mg·L⁻¹; copper (Cu) < 0.01 mg·L⁻¹; zinc (Zn) 0.01 mg·L⁻¹; sulfates (SO₄) 318 mg·L⁻¹; phosphate (PO₄) 38.5 mg·L⁻¹; nitrogen nitrate (NO₃⁻) 33.60 mg·L⁻¹; nitrogen nitrite (NO₂⁻) < 0.25 mg·L⁻¹; total dissolved solids (TDS—7.91 g·L⁻¹). Hence, the RDC was utilized as a substrate for three consecutive cultivation cycles (RDCs 1, 2, and 3), employing the same culture medium and microalgae with no additional nutrients or supplementation. A control experiment was conducted concurrently with the standard Zarrouk culture medium prepared in fresh water. In the control experiment, three consecutive cultivation cycles were also carried out (Controls 1, 2, and 3) using the same culture medium and microalgae without adding nutrients (Figure 1).

Cultivation was carried out in a raceway system (2.20 × 0.90 × 0.35 m) with 210 L of useful volume and with neither temperature nor light control for 30 days. In the region where the experiments were carried out, the average temperature and luminosity were 29–34 °C and 476–1784 μmol_photon m⁻²s⁻¹, respectively [14]. All cultures were agitated using submerged pumps 24 h per day for 30 days, with replacement of the evaporated water. The inoculum used in the experiments was the result of the previous cycle after being centrifuged (15,200× g for 3 min at 4 °C), and its biomass separated from the culture medium until reaching a concentration of approximately 0.5 g·L⁻¹. After reaching this concentration, a new cultivation cycle was started with the RDC with no addition of nutrients. At the end of each cultivation cycle, the biomass intended for subsequent analyses was separated by centrifugation (15,200× g for 3 min at 4 °C), washed with distilled water, and centrifuged again (15,200× g for 3 min at 4 °C). Analysis of the TDS of the supernatant in RDC 3 was carried out following the standard method for testing water and effluents, SM 2540C (APHA, 2005).

2.3. Biomass Concentration and pH

The biomass concentration (X) of Spirulina sp. LEB 18 was assessed every 24 h by measuring the optical density at wavelength λ of 670 nm using a digital spectrophotometer (PerkinElmer, Lambda 35 ultraviolet/visible spectrophotometer, Markham, ON, Canada) with a standard curve specific to Spirulina sp. LEB 18 (0.8722 × OD₆₇₀ − 0.2385). In addition, the pH levels were monitored daily using a digital pH meter (Sanxin PHS-3D pH meter, Shanghai, China).

2.4. Determination of Growth Parameters

From the biomass concentration values, the maximum biomass concentration (X_max in g·L⁻¹), maximum biomass productivity (P_max in g·L⁻¹·d⁻¹), final biomass productivity (P_final in g·L⁻¹·d⁻¹), and maximum specific growth (μ_max in d⁻¹) were calculated. The biomass productivity was determined as P_X = (X_t − X₀)/(t − t₀), where X_t is the biomass concentration (g·L⁻¹) at time t (in days, d), and X₀ is the biomass concentration (g·L⁻¹) at time t₀ (d). In addition, μ_max (d⁻¹) was determined by linear regression applied to the logarithmic phase of growth in a profile (ln X according to t), where the slope of the straight line was considered as the value of μ_max.

2.5. Recovery and Biochemical Characterization of Biomass

The biomass produced at the end of the experiments was recovered by centrifugation (Hitachi Himac CR-GIII, Tokyo, Japan) at 15,200× g and 4 °C for 3 min and then resuspended in distilled water. This procedure was repeated twice to eliminate any salts that might have adhered to the cells. The concentrated microalgal biomass was then frozen (at −80 °C), freeze-dried for (48 h), and stored (at −20 °C) until characterization [15].

The total protein content was assessed following the method outlined in [16], which involved thermal and alkaline pretreatment of the microalgal biomass. The determination of the total carbohydrate content followed the methodology proposed in [17], utilizing a glucose standard curve. The total lipid content was determined according to the method described in [18], which involved the extraction of nonpolar and polar lipids (at room temperature) using chloroform:methanol (2:1) and methanol:water (2:1), respectively. The analysis of chlorophylls a and b, as well as the total carotenoids (including carotenes and xanthophylls), was conducted simultaneously, utilizing the calculation described in [19]. The total phycocyanin content in the samples was determined according to the method outlined in [20].

2.6. Quantification and Determination of Amino Acids

The determination of the amino acids present in the Spirulina sp. LEB 18 biomass was conducted using the method proposed in [21]. Alkaline hydrolysis (4M KOH) was performed to determine the tryptophan content and acid hydrolysis (6M HCl at 110 °C for 22 h), which would in turn determine the remaining amino acids. Norvaline (2 mgmL⁻¹) was used as an internal standard. The free and total amino acid profiles were analyzed using an integrated system (Jasco, Tokyo, Japan) comprising an LC-NetII/ADC hardware interface, binary pump (Jasco PU-980), autosampler (Jasco AS-4150 RHPLC), multiwavelength detector (Jasco MD-2015 Plus), fluorescence detector (Jasco FP-2020 Plus), and oven (Model 7981, Hengoed, UK). Amino acid separation was performed on a ZORBAX Eclipse Plus C18 column (4.6 × 250 mm, 5 μm) from Agilent Technologies (Santa Clara, CA, USA—at 40 °C). The solvent gradient system, prepared in [22], comprised A) phosphate/borate buffer (10 mM Na₂HPO₄: 10 mM Na₂B₂O₇ (pH = 8.2): 5 mM NaN₃) and B) MeOH:ACN:H₂O (45:45:10, v/v/v). The gradient profile was set as follows: 0.85 min, 2% B; 33.4 min, 57% B; 33.5 min, 85% B; 39.3 min, 85% B; 39.4 min, 2% B; 40.0 min, 2% B (flow rate of 1.5 mLmin⁻¹). Fluorescence detection was monitored at λexcitation = 340 nm and λemission = 450 nm (from 0.0 to 26.2 min). Precolumn automatic derivatization in [22] combining two derivatization reagents, o-phthalaldehyde/3-mercaptopropionic acid (OPA/3-MPA) and 9-fluorenylmethyl chloroformate (FMOC), was adapted to the high-performance autosampler AS-4150 RHPLC from Jasco (Tokyo, Japan). All reagents used in the derivatization reaction were placed inside the autosampler in independent vials containing (i) 0.4 N borate buffer (pH = 10.2), (ii) OPA/3-MPA, (iii) FMOC, (iv) ultrapure water for sample dilution, and (v) ultrapure water for needle washing in-between reagents. For the OPA and FMOC derivatives, λ excitation = 266 nm and λ emission = 305 nm (from 26.2 to 40.0 min). Simultaneously, the OPA and FMOC derivatives were monitored at 338 and 262 nm, respectively.

2.7. Fatty Acid Methyl Ester Composition

The fatty acid methyl ester (FAME) composition and quantification were determined by transmethylation with boron trifluoride in hexane, followed by gas chromatography (DB-FFAP 30 m × 0.25 mm × 0.25 μm) with a flame ionization detector (GC-FID Clarus 680; Perkin–Elmer, Waltham, MA, USA). Injections of 1 μL were performed in split mode (1:50) and in triplicate. The temperatures for the injector and detector were set to 250 °C and 280 °C, respectively. The temperature started at 60 °C for 0.5 min, increased to 194 °C at a rate of 25 °C·min⁻¹, and was maintained for 1 min. Subsequently, there was a further increase to 235 °C at 5 °C·min⁻¹, followed by a minute-long maintenance. FAMEs were identified by comparing their retention times with those of the standard chromatogram (C4–C24, 189–19 AMP Sigma-Aldrich, St. Louis, MO, USA). The results were expressed in percentages, and the peak areas were determined according to the method proposed in [8] using the Clarus Chromatography software to normalize the percentage areas of total fatty acids.

2.8. Statistical Analysis

Statistical treatment was applied to all quantitative analyses. The results were analyzed using two statistical tests to compare and demonstrate the efficiency of the PW-supplemented treatments. One-way analysis variance and Tukey’s test at a 95% confidence level were conducted using the Statistica software (version 10.0) to compare the means. The Shapiro–Wilk test was applied to assess the normality of data. The experimental results were expressed as the means ± standard deviations of triplicate experiments.

3. Results and Discussion

3.1. Growth Parameters

In [10], using 100% DC supplemented with 25% of Zarrouk medium nutrients, showed promise as a culture medium for Spirulina sp. LEB 18 across the three recycling stages (

Table 1. Average results of maximum biomass concentration (X_max, g·L⁻¹), final biomass concentration (X_final, g·L⁻¹), maximum biomass productivity (P_max, g·L⁻¹·d⁻¹), final biomass productivity (P_final, g·L⁻¹·d⁻¹), and specific growth (µ_max, d⁻¹) of Spirulina sp. LEB 18 grown in reused culture medium from the study proposed in [10].

	Assays	Xmax (g·L−1)	Xfinal (g·L−1)	Pmax (g·L−1·d−1)	Pfinal (g·L−1·d−1)	µmax (d−1)	pH
Mata et al. (2020) [10]	Control	-	1.25 ± 0.01	-	0.03 ± 0.02	0.02 ± 0.00	10.15 ± 0.12
	DC	-	1.14 ± 0.01	-	0.05 ± 0.01	0.03 ± 0.00	9.75 ± 0.52
Recycle 1	Control 1	1.21(30th) ± 0.24 cA	1.20(30th) ± 0.02 bA	0.04 ± 0.01 dA	0.04 ± 0.01 dA	0.02 ± <0.01 bA	10.50 ± 0.17 aA
	RDC 1 *	1.24(29th) ± 0.15 bA	1.13(30th) ± 0.05 cB	0.04 ± <0.01 cA	0.03 ± 0.01 cB	0.02 ± <0.01 bA	10.36 ± 0.24 cB
Recycle 2	Control 2	0.88(9th) ± 0.17 dB	0.54(27th) ± 0.02 eB	0.08 ± 0.02 eA	0.01 ± 0.00 eB	0.02 ± <0.01 bA	10.45 ± 0.07 bA
	RDC 2 *	1.28(19th) ± 0.19 bA	0.71(28th) ± 0.15 dB	0.05 ± 0.01 bA	0.01 ± 0.01 dB	0.01 ±< 0.01 bA	10.23 ± 0.11 dB
Recycle 3	Control 3	1.36(26th) ± 0.25 aA	1.35(26th) ± 0.21 aA	0.04 ± 0.02 aA	0.04 ± 0.02 aA	0.03 ± <0.01 aA	9.50 ± 0.14 eA
	RDC 3 *	1.25 (12th) ± 0.33 bB	0.57(24th) ± 0.17 eB	0.09 ± <0.01 bA	0.01 ± <0.01 eB	0.02 ± <0.01 bA	9.47 ± 0.25 fB
ΣTotal	Control	3.45 B	3.09 A	0.371 A	0.06	-	-
	RDCs	3.77 A	2.41 B	0.703 B	-	-	-

Lowercase letters with equal superscripts in the same column, for the same parameter, indicate that the means do not show a statistical difference at the 95% confidence level between the cycles (p > 0.05). Capital letters with equal superscripts in the same column, for the same parameter, indicate that the means do not show a statistical difference at the 95% confidence level in the same cycle (p > 0.05). Control: test carried out in standard Zarrouk medium in fresh water; RDCs 1, 2, and 3 *: first, second, and third reuses of cultivation medium containing DC, respectively. Σ Total control and RDCs: Sum of total concentrations of the control assays and reused media, respectively.

). Also in [10], a maximum production (X_max) of 1.14 g·L⁻¹ was reached on the 24th day of cultivation of Spirulina sp. LEB 18 in DC supplemented with 25% of Zarrouk medium nutrients without recycling (Table 1). In the present study, higher values for this parameter were observed, of which RDC 3 reached X_max of 1.25 g·L⁻¹ in less time (12th day) than both RDC 2 with 1.28 g·L⁻¹ on the 19th day and RDC 1 with 1.24 g·L⁻¹ on the 29th day (Table 1). Remarkably, in RDC 3, the maximum and final concentration occurred in a shorter cultivation time compared with the other recycles, possibly due to the depletion of nutrients, which served as support to enable cell growth in RDCs 1 and 2. Therefore, the cellular production found in this study demonstrates the potential of Spirulina sp. LEB 18 to reach relevant quantities of biomass using medium recycling up to three successive times.

Evaluating the treatments in relation to their corresponding controls, a significant reduction (p > 0.05) of X_max in RDC 3 was observed by only 8% and X_final by 58% (Table 1). This reduction in Spirulina sp. LEB 18 growth may be associated with low nitrogen concentrations and/or phosphorous limitation. This limitation originated from the setup in the previous study, in which phosphate (PO₄) was 83.11% removed, while nitrate (NO₃⁻) was 96.99% removed and nitrite (NO₂⁻) was not detected [10]. Nevertheless, nutrient limitation did not hinder the production of microalgal biomass in RDC 3 until the 12th day of cultivation. Accordingly, after this period, strategies such as nutritional supplementation or semicontinuous cultivation may maximize the biomass and metabolite production [23,24].

RDC 2 resulted in an X_max 45% higher than its control test. At the end of the cultivation, RDC 1 resulted in an X_final only 5.8% lower than the control condition, unlike RDC 2 that promoted an X_final 24% higher than its corresponding control. Furthermore, in all recycling stages, specific growth (µ_max) was observed with similar values (0.01–0.03 d⁻¹) (p < 0.05) to their controls. The cultivation of Spirulina sp. LEB 18 using DC without recycling, as performed in [10], resulted in a final biomass production reaching 1.14 g·L⁻¹ (Table 1). By reusing this same cultivation medium up to three times, a final microalgae production of 2.41 g·L⁻¹ was achieved along with X_max of up to 3.77 g·L⁻¹ (Table 1).

The pH values found in the study suitably agreed with those considered ideal for the growth of Spirulina (9.5–10.5) and ranged from 9.47 (RDC 3) to 10.50 (control 1). The cultivation of Spirulina sp. LEB 18 using DC without recycling, as indicated by these values, not only reduces the likelihood of contamination by other microalgal species but also does not impact the growth, pigment production, and protein content of Spirulina [8,25]. Additionally, the average temperature of the bioreactors during experiments varied from 27.70 °C (minimum) to 42.80 °C (maximum), with an average of 31.22 ± 1.37 °C. Thus, it did not negatively affect the development of microalgae because they are thermophilic species with excellent growth between 35 °C and 37 °C [26].

Biodesalination promoted by Spirulina sp. LEB18 in the recycles achieved a removal rate of 74.64% in RDC 3, having 7.91 g·L⁻¹ of initial TDS and reaching 0.05 g·L⁻¹ in the last cycle. We believe that TDS and cellular energy reserve compounds maintained cell growth, because the nitrogen (N) and phosphorus (P) sources were depleted primarily during RDC cultivation. TDS are composed of ions and dissolved organic material, which serve as important resources for mixotrophic microalgae, such as Spirulina sp. LEB 18, for absorption and/or consumption [27,28]. A similar result was observed in [29] using Arthrospira and Dunaliella microalgae in saline aquaculture-effluent biodesalination. The authors observed that both species completely removed phosphate and approximately 75% of the total nitrogen from the medium, followed by approximately 33% TDS removal from 21,000 to 14,000 mg·L⁻¹. Thus, the set of growth parameters evaluated allowed us to highlight the difference in this study for the production of Spirulina sp. LEB 18 biomass, reused in up to three DC cycles, without requiring additional nutrient supplementation in an open system at high temperatures. The implementation of a microalgae cultivation strategy was promoted with lower nutrient requirements, showing that Spirulina biomass can be obtained in concentrations higher or at least similar to those of cultivation systems that demand greater nutritional inputs and fresh water.

3.2. Determination of Chlorophylls A and B, Total Carotenoids, and Phycocyanin

RDCs 2 and 3 showed a greater significant reduction (p < 0.05) of all photosynthetic pigments present in the biomass of Spirulina sp. LEB 18 (Figure 2). The degradation or decrease in photosynthetic pigments like chlorophyll a and b serves as a response to stress in microalgae [30]. In situations of oxidative stress, reactive oxygen species (ROS) are generated and built up within microalgae cells, leading to the degradation of the PS II complex of photosynthesis and a reduction in their photosynthetic efficiency [31]. The findings in [10] reinforce this effect of stress on the concentration of pigments, given a significant reduction in all pigments observed in relation to the control. The sharp pigment reduction in RDCs 1, 2, and 3 may be related to a metabolic change called chlorosis, which is the ability of microalgae to direct their metabolism to use chlorophyll as an intracellular source of nitrogen, consequently reducing the synthesis of this pigment [32]. Another factor may be related to the decrease in phosphorus in crops. For instance, limited phosphorus (0.01 g·L⁻¹ of K₂HPO₄) in the Zarrouk medium has resulted in a reduction of 61% in the total chlorophyll content in Spirulina platensis biomass compared with the control [33].

The fluctuations and reduction in carotenoid and phycocyanin concentrations (p < 0.05) (Figure 2) may be attributed to osmotic regulation, because microalgal cells present different responses against variations in nutrients, mainly N, P, and Fe/Na, resulting in changes in photosynthetic activity throughout the crop [34,35]. In [36], reductions in the content of chlorophyll (61.4%) and carotenoids (28.4%) were observed in Spirulina maxima biomass when cultivated in seawater (total salinity of 35 g·L⁻¹) supplemented with carbon (0.2 g·L⁻¹ of NaHCO₃), nitrogen (2.0 g·L⁻¹ of NaNO₃), phosphorus (0.016 g·L⁻¹ of K₂HPO₄), and iron (0.01 g·L⁻¹ of ferric ethylenediaminetetraacetic acid) compared with the control (fresh water in Spirulina–Ogawa–Terui medium).

Intense light can stimulate the protection mechanism against photooxidation and reduce the accumulation of photosynthetic pigments (chlorophylls and phycobiliproteins) [37]. Intense light in the experimental region may have compromised the synthesis of phycocyanin. In [10], a reduction in phycocyanin of up to 74% was observed in the Spirulina sp. LEB 18 biomass cultivated (30 d) using DC without recycling. The authors attributed this finding to the cultivation being carried out in the Northeast region of Brazil, which is exposed to high luminosity and solar radiation (5–8 h, mean of 7 h), conditions similar to those in this study. In addition to this factor, the recycling strategy used in this study probably promoted stress and an inhibitory effect on the microalgae’s photosystem, because with each use of the recycled medium, the content of the main nutrients (i.e., N and P) necessary for photosynthesis was reduced by small quantities.

3.3. Biomass Biochemical Composition

Regarding the total protein content of Spirulina sp. LEB 18, cultures with RDC presented lower contents than their corresponding controls, with reductions in proteins up to 48.5% (RDC 1), 51.9% (RDC 2), and 47.5% (RDC 3) after each cycle. However, even under reduction, the first recycle (RDC 1) presented a concentration for this macromolecule in the microalgae biomass of up to approximately 40% (ww⁻¹—weight/weight (dry weight)) (Figure 3). When cultivating Spirulina sp. LEB 18 using DC without recycling, a similar reduction in protein content (~26%) was observed in the final microalgae biomass [10]. Osmotic and ionic stress induced by the elevated total salinity of the environment, combined with the depletion of nutrients such as nitrogen, may have triggered an adaptive process of cellular osmoregulation, demanding energy to maintain metabolism and secondary synthesis of low molecular weight carbohydrates to the detriment of protein synthesis.

The same behavior was observed for lipid content in the present study, with a reduction of this metabolite after each recycle by up to 22.6% (RDC 1), 9.61% (RDC 2), and 45.4% (RDC 3) compared with the corresponding controls (Figure 3). However, the production of 9.2% and 9.4% of this molecule was observed in RDCs 1 and 2, respectively, which were 22.6% and 9.6% lower than the corresponding controls. The reduction in the total lipid content in these cultures can be attributed to the reduction in nutrients (mainly N and P) with each recycle, owing to a higher total salinity (Na⁺) in the medium compared with the control conditions. These factors may promote a shift in metabolic energy toward the production of carbohydrates to maintain the microalgal intra and extracellular osmoregulatory balance. Moreover, this stressful condition could have triggered the production of ROS, leading to cellular damage and lipid peroxidation [38].

In the present study, RDC 1 showed accumulation of carbohydrate content by up to 67% compared with the corresponding control (Figure 3). Subsequent recycling of this medium promoted the reduction of this macromolecule compared with the control condition. However, under individual observation of each recycling stage, carbohydrate contents of 12.5% ww⁻¹ in RDC 2 and 16.9% ww⁻¹ in RDC 3 were obtained in the final Spirulina biomass. These results are likely due to ionic/osmotic stress caused by the reduction/deprivation of nitrogen and phosphorus sources in the extracellular environment [9,10,39].

3.4. Quantification and Determination of Amino Acids

Regarding the amino acid profile, this study observed a decrease (p > 0.05) in the concentrations of both essential and non-essential amino acids in the Spirulina sp. LEB 18 biomass cultured in each RDC cycle (

Table 2. Amino acid profile (mgg⁻¹ biomass) of Spirulina sp. LEB 18 grown in reused culture medium containing desalination concentrate (DC) and 25% Zarrouk components.

Amino Acid	Control 1	RDC1	Control 2	RDC2	Control 3	RDC3
Aspartic acid	43.98 ± 2.35 a	19.25 ± 1.02 b	18.80 ± 0.40 bc	11.53 ± 0.31 d	17.54 ± 0.57 c	11.35 ± 0.38 d
Glutamic acid	56.81 ± 2.92 a	25.05 ± 1.30 b	24.56 ± 0.52 b	14.87 ± 0.41 d	21.56 ± 0.66 c	14.70 ± 0.44 d
Serine	22.80 ± 1.18 a	10.05 ± 0.52 b	7.71 ± 0.16 c	5.15 ± 0.16 d	8.01 ± 0.26 c	5.08 ± 0.16 d
Histidine	7.49 ± 0.35 a	2.61 ± 0.13 b	2.64 ±0.05 b	1.40 ± 0.07 c	2.26 ± 0.07 b	1.38 ± 0.08 c
Glycin	26.63 ± 1.36 a	11.62 ± 0.68 bc	10.04 ± 0.21 c	7.31 ± 0.20 d	10.84 ± 0.40 c	7.25 ± 0.21 d
Threoninae *	19.89 ± 0.97 a	8.71 ± 0.42 b	7.45 ± 0.13 bc	5.27 ± 0.14 d	6.87 ± 0.21 c	5.21 ± 0.14 d
Arginine	36.46 ± 1.81 a	15.25 ± 0.91 b	15.25 ± 0.51 b	8.54 ± 0.31 d	12.86 ± 0.24 c	8.35 ± 0.41 d
Alanine	36.73 ± 1.71 a	15.92 ± 0.83 b	13.11 ± 0.32 c	9.06 ± 0.25 d	13.77 ± 0.43 c	8.93 ± 0.27 d
Tirosine	19.91 ± 1.15 a	7.74 ± 0.36 b	5.07 ± 0.13 c	3.36 ± 0.13 d	2.60 ± 0.14 e	3.23 ± 0.29 d
Valine *	26.69 ± 1.35 a	11.66 ± 0.62 b	10.28 ± 0.26 b	6.87 ± 0.16 c	10.65 ± 0.31 b	6.70 ± 0.22 c
Methionine *	9.26 ± 0.50 a	3.82 ± 0.38 b	2.15 ± 0.06 c	1.42 ± 0.06 d	1.08 ± 0.06 d	1.26 ± 0.14 d
Tryptophan *	2.48 ±0.18 a	0.84 ± 0.11 b	0.85 ± 0.04 b	0.50 ± 0.01 c	0.87 ± 0.06 b	0.49 ± 0.02 c
Fenilalanine *	26.82 ±1.31 a	11.14 ± 0.55 b	9.78 ± 0.28 c	6.67 ± 0.18 d	10.19 ± 0.30 b	6.51 ± 0.22 d
Isoleucine *	22.37 ± 1.12 a	9.44 ± 0.47 b	7.86 ± 0.19 d	5.14 ± 0.12 e	8.67 ± 0.26 c	5.06 ± 0.14 e
Leucine *	48.70 ± 2.38 a	20.87 ± 0.98 b	17.54 ± 0.42 d	11.66 ± 0.29 e	18.62 ± 0.55 c	11.48 ± 0.32 e
Lysine *	21.71 ± 1.01 a	9.88 ± 0.43 b	7.48 ± 0.07 c	5.56 ± 0.16 d	7.60 ± 0.22 c	5.61 ± 0.12 d
Hidroxiproline	0.32 ± 0.01 a	0.18 ± <0.01 b	0.17 ± <0.01 b	0.18 ± <0.01 b	0.17 ± <0.01 b	0.18 ± <0.01 b
Proline	17.06 ± 0.74 a	7.48 ± 0.34 b	6.32 ± 0.20 c	4.56 ± 0.11 d	6.69 ± 0.17 c	4.47 ± 0.16 d
Essentials	177.92 a	76.36 b	63.38 c	43.07 d	64.54 c	42.30 d
Non essentials	268.19 a	115.14 b	103.67 c	65.96 e	96.29 d	64.90 e
Total	446.10 a	191.51 b	167.06 c	109.04 d	160.83 c	107.21 d

Equal superscript lowercase letters in the same column, for the same parameter, indicate that the means do not show statistical difference at the 95% confidence level (p > 0.05). Control: test carried out in standard Zarrouk medium in fresh water; RDCs 1, 2, and 3: first, second and third reuses of cultivation medium containing DC, respectively. Essential amino acids. The results are shown as means ± standard deviations of the concentrations of amino acids present in the microalgae biomass on the last day of cultivation.

). This reduction may be related to the reduction in the nitrogen source with each cycle. In the synthesis of primary amino acids like glutamic acid, nitrogen is assimilated into carbon structures [40]. These synthesized amino acids serve as N donors for the primary N compounds found in photosynthetic microorganisms. Consequently, the depletion of the nitrogen source compromises amino acid synthesis.

Despite the decrease in amino acid content, Spirulina sp. LEB 18 biomass contained all essential amino acids (i.e., threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and lysine), constituting approximately 40% ww⁻¹ of the total proteins (Table 2). According to the Food and Agriculture Organization and World Health Organization, the produced biomass demonstrates its potential as a source of essential amino acids and can be considered for inclusion in an ”ideal diet” [41].

The major amino acids in all recycles were aspartic acid, glutamic acid, and leucine (Table 2) [42]. Aspartic and glutamic acids are amino acids with large effects on the central nervous system, functioning as excitatory neurotransmitters. They play crucial roles in various physiological processes and health conditions, including memory, learning, and movement disorders. The balance of these amino acids plays a fundamental role in brain functions, and any alternation can lead to various neurological or psychiatric disorders. For instance, leucine plays a crucial role in the secondary structure of muscle protein, facilitating protein synthesis in the muscles [43,44]. Therefore, these amino acids should be present in the diet of humans and animals. In [10], the absence of heavy metals, such as lead, cadmium, nickel, and copper, was confirmed in the Spirulina sp. LEB 18 biomass grown using DC without reuse. This suggests that the biomass in all crops grown in the present study also flacked these metals, possibly enabling the inclusion in foods intended for animal consumption.

3.5. FAME Composition

Considering that control 1, RDC 1, control 2, and RDC 2 exhibited higher lipid concentrations than RDC 3 and showed no statistically significant difference between them (p > 0.05), they were selected to assess the profile and composition of free fatty acids. Because cyanobacteria have complex lipid structures within their cells, including phospholipids and glycolipids, most of their lipids are saponifiable, resulting in increased free fatty acids production (

Table 3. Fatty acid profile (%) of Spirulina sp. LEB 18 grown in reused culture medium containing desalination concentrate (DC) and 25% Zarrouk components.

Treatments
Fatty Acid	Nomenclature	Control 1	RDC1	Control 2	RDC2
C10:0	Capric acid	5.78 ± 0.15 b	6.46 ± 0.69 a	4.27 ± 0.05 c	3.51 ± 0.04 d
C12:0	Lauric acid	1.35 ± 0.29	-	-	-
C13:0	Tridecylic acid	1.84 ± 0.02	-	-	-
C15:0	Pentadecylic acid	0.97 ± 0.01	-	-	-
C15:1	Pentadecenoic acid	44.5 ± 0.14 c	42.96 ± 0.42 d	50.23 ± 0.40 a	48.87 ± 0.18 b
C16:0	Palmitic acid	3.06 ± 0.02 b	2.89 ± 0.20 c	7.12 ± 0.07 a	7.62 ± 0.05 a
C17:1	Heptadecanoic acid	1.9 ± 0.13 d	3.58 ± 0.39 c	6.59 ± 0.37 b	7.80 ± 0.07 a
C18:0	Stearic acid	6.86 ± 0.31 b	8.93 ± 2.30 a	3.11 ± 0.05 c	3.45 ± 0.44 c
C18:1n9c	Oleic acid	14.5 ± 0.03 b	13.89 ± 0.37 c	15.19 ± 0.57 b	17.17 ± 0.07 a
C18:2n6t	Linolelaidic acid	19.24 ± 0.10 a	18.03 ± 0.98 b	13.49 ± 0.15 c	11.57 ± 0.03 d
C18:3n6	α-linolenic acid	-	3.25 ± 0.36	-	-
Σ saturated		19.86	18.28	14.49	14.58
Σ monounsaturated		60.90	78.72	72.01	73.84
Σ polyunsaturated		19.24	21.29	13.49	11.57

Equal superscript lowercase letters in the same column, for the same parameter, indicate that the means do not show a statistical difference at the 95% confidence level (p > 0.05). Control: test carried out in standard Zarrouk medium in freshwater; RDCs 1, 2, and 3: first, second, and third reuses of the cultivation medium containing DC, respectively. The results are shown as means ± standard deviations of the concentrations of fatty acids present in the microalgae biomass on the last day of cultivation.

). The control and RDC cycles showed elevated levels of monounsaturated fatty acids, with the highest values observed in RDCs 1 and 2 attributed to the high salinity of the saline concentrate inducing osmotic stress, which affects the membrane fluidity and permeability. To counteract these effects, monounsaturated fatty acids are synthesized to maintain the cell membrane fluidity and prevent damage. In the control group, the increase in monounsaturated content is linked to oxidative processes that require O₂, the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, as well as substrate that serve to mitigate the formation of ROS under stress conditions induced by consecutive nutrient shortages [10,45].

The main fatty acid was pentadecanoic acid (C15:1) in the control and RDC. Microalgae cultures are expected to synthesize fatty acids from the C16:0 or C18:0 families in high concentrations, which may undergo changes in desaturase and elongase enzymes, resulting in diverse unsaturated and polyunsaturated fatty acids. Furthermore, these metabolic changes may be the result of a possible combination of environmental stresses (e.g., temperature, light, and season) exerted on crops in outdoor systems. The same result was observed in [10] under the same cultivation conditions, where C15:1 was the main fatty acid in the control (41.83%) and in the cultivation with saline concentrate (39.68%). Therefore, a change in the metabolism of Spirulina sp. LEB 18 was observed, resulting in the production of additional monounsaturated and polyunsaturated fatty acids. The order of the major fatty acids followed an alternation between oleic acid (C18:1n9c) and linolelaidic acid (C18:2n6t) between the cycles. The synthesis of C18:2n6t is related to the protection mechanism against high exposure to solar irradiation and hyperosmotic stress. The synthesis and accumulation of C18:1n9c is interesting for introducing microalgal biomass into food because its consumption has been related to lower levels of total cholesterol and low-density lipoprotein cholesterol, an improved glycemic profile, the prevention of some types of cancer, and the improved stability of biomass against lipid oxidation, which negatively affects the organoleptic and sensorial attributes of the product [46].

4. Conclusions

This study successfully demonstrates the viability of using RDC as a culture medium for the outdoor cultivation of Spirulina sp. LEB 18. Reusing up to three cultivation cycles of Spirulina sp. LEB 18, using 100% DC containing 25% of the components of the Zarrouk medium, promoted a total biomass production (3.77 g·L⁻¹) above that using the synthetic medium (Zarrouk). The resulting biomass showed a balanced biochemical composition between protein, carbohydrates, and lipids, with emphasis on the protein composition (≤37%), including all essential amino acids (i.e., threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and lysine), constituting approximately 40% of the total proteins and fatty acid as C18:1n9c ideal for inclusion in foods. Overall, this study introduces an innovative approach to biomass production with the potential of RDC as an alternative medium, achieving concentrations comparable to or higher than standard systems requiring greater nutritional inputs and fresh water.