Synergistic Effects of Light and Salinity on Carotenoid and Biomass Composition of Synechocystis PCC6803 Cultures

Abstract

Light and salt stress affect the growth of plants and microorganisms, causing photo-oxidative stress. The cyanobacterium Synechocystis is notable for its adaptability to and sustainability in seawater. In this study, the synergistic effects of different light intensities and salt concentrations on the growth and biomass composition of Synechocystis were examined. Cultures were grown in BG11 medium (control) and with 20 and 40 g L⁻¹ marine salts (obtained from a commercial sea water preparation) at 100, 200, and 400 μmoles photons m⁻² s⁻¹ (LL, ML, and HL, respectively) to assess the interactive effects of salinity stress and increasing light intensity. The effect of salinity stress was most pronounced under LL and ML, where the highest accumulation of all major carotenoids was observed; under HL, the contents of most carotenoids significantly increased mainly at the highest salt concentration but to a lesser extent). Under LL and ML echinenone reached the highest values (2.71-fold and 3.75-fold higher than in the control, respectively), whereas β-carotene showed the highest increase at LL, reaching concentrations three times those of the control. At HL myxoxanthophyll exhibited the highest increase with marine salt (1.9-fold higher than in the control). The results show that Synechocystis could grow at all light intensities and marine salt concentrations via increased synthesis of carotenoids in response to physiological stress.

1. Introduction

Light and salt stress are two major environmental factors that limit plant development and production, as well as the growth of photosynthetic microorganisms [1,2,3]. Exposure of photosynthetic organisms to high light intensity reduces the activity of the photosynthetic apparatus, leading to a decline in photosystem II (PSII) quantum yield, due to photo-oxidative stress. Several studies have investigated the induction of carotenoid synthesis under photo-oxidative stress and other conditions that promote the formation of reactive oxygen species (ROS) [4,5,6,7,8].

In natural environments, salt stress often coexists with light stress. Numerous studies have examined the effects of salt stress on PSII under light stress [9,10,11,12,13]. Salt stress, one of the most prevalent abiotic stressors in nature, involves both osmotic and ionic stress. Osmotic stress and high cellular ion concentrations can affect normal metabolic activities and photosynthesis. Furthermore, salt stress increases ROS levels within cells. Excessive ROS inhibits photosynthesis by damaging the photosynthetic machinery, preventing the production of proteins associated with the photosystem, such as the D1 protein, and degrading the structure of the thylakoid membrane. It has been proposed that, in the cyanobacterium Synechocystis, photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII primarily by suppressing de novo protein synthesis [14]. Cyanobacteria have developed specific metabolic processes and regulatory systems to cope with changing environments [15]. For example, the unicellular freshwater cyanobacterium Synechocystis sp. PCC6803 can withstand up to 1.2 M NaCl [16]. Cyanobacteria are considered model organisms for studying photosynthetic responses to fluctuations in growth conditions [17] and salt stress [9,18] because of their rapid and quantifiable physiological responsiveness and their photosynthetic machinery being structurally and functionally homologous to the chloroplasts of eukaryotic cells [19,20]. In microalgae (eukaryotic cells), photosynthesis occurs within membrane-bound chloroplasts, and thylakoids are generally organised into stacks (grana). In cyanobacteria (prokaryotic cells), compartmentation is absent (no chloroplast), and thylakoids are organised in membrane layers within the cytoplasm. Whereas microalgae use chlorophyll–carotenoid–protein complexes as light–harvesting complexes, cyanobacteria use phycobilisomes, such as phycocyanin and phycoerythrin.

Interestingly, in previous studies [21,22], salt stress in the cyanobacterium Limnospira platensis did not directly affect PSII activity in the dark. However, when combined with exposure to different light intensities, photosynthetic activity was impaired, with the degree of inhibition proportional to the light intensity.

When the photosynthetic system is under stress (nutrient limitation or starvation, excessive light, salt stress, or high or low temperature), it may not be able to use the absorbed light energy efficiently, thereby promoting dissipation processes. When photosynthetic microalgal cells receive a signal indicating the induction of a highly reductive state, defensive mechanisms are activated to quench the accumulated electrons. Carotenoids play a critical role in protecting the photosynthetic apparatus from photo-oxidative damage. They quench singlet oxygen and dissipate excess energy as heat, preventing damage to the photosystems.

Carotenoids are essential pigments in cyanobacteria, including Synechocystis, and they play crucial roles in photosynthesis and photoprotection. The carotenoids synthesised include lutein, the xanthophyll cycle pigments violaxanthin, antheraxanthin, and zeaxanthin, as well as loroxanthin and fucoxanthin [23,24,25].

Carotenoids in Synechocystis are synthesised through the methylerythritol phosphate (MEP) pathway. The process begins with the synthesis of phytoene, which is then converted through several desaturation steps into lycopene. Lycopene serves as a branch point for the generation of various carotenoids, including β-carotene, zeaxanthin, and echinenone [26].

The synthesis of carotenoids in Synechocystis is influenced by various environmental factors. Among these, salt stress induces carotenoid synthesis in Synechocystis, enhancing the organism’s ability to cope with oxidative stress and maintain photosynthetic efficiency.

Under salt stress, genes involved in carotenoid biosynthesis, such as crtB, crtP, and crtQ, are upregulated. This leads to an increased production of carotenoids such as β-carotene, zeaxanthin, and echinenone. Synechocystis, a versatile cyanobacterium, has several potential applications in various industries due to its unique properties and capabilities. Enhanced carotenoid production has significant implications for industrial applications, including the production of natural colourants, antioxidants, and nutritional supplements in the pharmaceutical, nutraceutical, cosmetics, and food industries. The high contents of carotenoids, including zeaxanthin and echinenone, can be extracted and used as natural antioxidants in pharmaceuticals and nutraceuticals [27,28]. Carotenoids and other pigments from Synechocystis can be used in skin care products for their antioxidant and UV-protective properties. The antioxidant properties of carotenoids help reduce oxidative stress, making them valuable in anti-ageing formulations. Pigments such as phycocyanin and carotenoids can be used as natural colourants in food products.

The ability of Synechocystis to fix CO₂ makes the cultivation of this cyanobacterium important for helping to mitigate climate change. Moreover, the possibility of growing the organism in seawater makes its cultivation relevant from a sustainability perspective. The combination of producing biomass enriched with carotenoids and using the same growth conditions for the concomitant production of biofuels, such as hydrogen, makes the cultivation of Synechocystis with marine salts highly suitable for producing value-added biomass and sustainable energy [29].

Synechocystis is extensively studied as a model organism for photobiological hydrogen production; however, information on the combination of light and salinity, two environmental factors that can significantly affect biomass composition, is very limited. Outdoor cultures are usually exposed to additional environmental stresses, in combination with light, which can synergistically affect both growth and cell composition [11,18]. The use of salinity for growing Synechocystis outdoors is important to prevent grazing by competing organisms [29] and to stimulate carotenoid synthesis for protecting the photosynthetic apparatus, resulting in an enrichment of these valuable compounds in the biomass [30,31]. These considerations prompted us to investigate the effects on the growth and carotenoid composition of Synechocystis cells exposed to a combination of light and salinity.

This information is important for mass culture in seawater, given the need to improve process sustainability. The tested salinities are both lower and higher than seawater values and can help explain the culture’s behaviour during strong dilution from rainfall or when evaporation is offset by adding marine water.

The purpose of using increasing light intensities and salt concentrations was to allow for more detailed monitoring of the effects of increasing stress on the growth and biochemical composition of cells and to determine whether the synergistic effect of salt and light is proportionally enhanced with increasing stress conditions.

2. Results

The results are reported for Synechocystis cultures grown at 100 and 400 μmoles photons m⁻² s⁻¹ to highlight the impact of different salinities at these intensities, considered as low- and high-light conditions, respectively (hereafter referred to as LL and HL). The effects on growth, photosynthetic activity, and biochemical composition are presented. Changes in the carotenoid profile are reported for Synechocystis cultures grown at three light intensities to highlight the synergistic effect of salinity stress with increasing light intensity: 100, 200 (medium light, ML), and 400 μmoles photons m⁻² s⁻¹, as these are two stress factors affecting both carotenoid content and biomass composition.

2.1. Changes in Growth

The results of the changes in biomass accumulation, measured as dry weight (DW), under LL and HL, are reported in Figure 1. The results demonstrate that Synechocystis could grow under both LL and HL in the presence of different marine salt concentrations. Biomass accumulation was higher under HL than under LL, indicating that the growth of the cultures increased when they were exposed to higher light intensity, even in the presence of marine salts.

At each light intensity, no significant differences were observed at the end of the growth, between the conditions, although under HL, the DW value of the BG11 culture was the highest, probably due to the salts interfering with growth.

2.2. Changes in Total Chlorophyll-a and Total Carotenoid Content

The chlorophyll-a concentration varied according to the salt concentrations and light intensities. The results are reported for LL and HL (Figure 2). The control culture in BG11, exposed to 100 μmoles photons m⁻² s⁻¹, showed the highest increase in chlorophyll-a (3.5 times higher), followed by the cultures supplemented with 20 g L⁻¹ and 40 g L⁻¹ marine salts (2.7- and 1.8-fold, respectively), compared to the initial values. At the end of growth, the chlorophyll-a contents significantly differed. The same result was obtained at a light intensity of 400 μmoles photons m⁻² s⁻¹. In the BG11 culture, the chlorophyll-a content was higher than in the cultures in seawater at the end of growth, but the differences were not significant.

Concerning the accumulation of total carotenoids at 100 μmoles photons m⁻² s⁻¹, the changes followed the same pattern observed for chlorophyll-a, with the highest increase detected in the BG11 culture and with 20 g L⁻¹ (2.4-fold higher), while in the presence of the higher marine salt concentration of 40 g L⁻¹, a lower increase was detected (2.0-fold compared to the initial value). Also, at a light intensity of 400 μmoles photons m⁻² s⁻¹, the total carotenoid content was higher in the BG11 culture than in the cultures in seawater at the end of growth, but the differences were not significant.

The changes in the carotenoid/chlorophyll (Car/Chl) ratio during growth under 100 and 400 μmoles photons m⁻² s⁻¹ are reported in

Table 1. Changes in the total carotenoid/chlorophyll (Car/Chl) ratio in Synechocystis sp. PCC6803 cultures grown at a light intensity of 100 μmoles photons m⁻² s⁻¹ in BG11 and in BG11 + 20 g L⁻¹ and BG11 + 40 g L⁻¹ marine salts. Different letters indicate significant differences (p < 0.05) between salinities. The statistical analysis refers exclusively to the final values.

Time (h)	BG11	BG11 + 20 g L−1	BG11 + 40 g L−1
0	0.526 ± 0.006	0.558 ± 0.008	0.442 ± 0.004
24	0.384 ± 0.006	0.414 ± 0.035	0.343 ± 0.014
48	0.356 ± 0.012	0.393 ± 0.012	0.315 ± 0.010
72	0.320 ± 0.013	0.367 ± 0.082	0.352 ± 0.107
96	0.349 ± 0.010 a	0.492 ± 0.022 b	0.513 ± 0.056 b

and

Table 2. Changes in the carotenoid/chlorophyll (Car/Chl) ratio (see Table 1) in Synechocystis sp. PCC6803 cultures grown at a light intensity of 400 μmoles photons m⁻² s⁻¹ in BG11 and in BG11 + 20 g L⁻¹ and BG11 + 40 g L⁻¹ marine salts. Different letters indicate significant differences (p < 0.05) between salinities. The statistical analysis refers exclusively to the final values.

Time (h)	BG11	BG11 + 20 g L−1	BG11 + 40 g L−1
0	0.443 ± 0.013	0.366 ± 0.002	0.381 ± 0.031
24	0.456 ± 0.008	0.443 ± 0.021	0.396 ± 0.003
48	0.272 ± 0.030	0.463 ± 0.044	0.432 ± 0.058
72	0.274 ± 0.040	0.376 ± 0.038	0.367 ± 0.020
96	0.299 ± 0.017 a	0.407 ± 0.011 b	0.340 ± 0.017 b

, respectively.

The largest changes were observed in the BG11 cultures, where the Car/Chl ratio decreased under both light intensities, declining relative to the initial values (Table 1). In the cultures with marine salts, under both LL and HL, the changes were less significant. As expected, the Car/Chl ratio reached a steady state much faster under higher light than under lower light (Table 2). At the beginning of the exposure period, the cultures acclimated to marine salts showed lower Car/Chl ratio values than the BG11 cultures under both LL and HL.

2.3. Carotenoid Profile

To gain further insight into the carotenoid changes in the Synechocystis cultures exposed to different salt concentrations, results from cultures grown under a light irradiance of 200 μmoles photons m⁻² s⁻¹, hereafter referred to as ML, are also reported.

Examining the changes in the carotenoid composition at the end of growth, a two-way ANOVA revealed an interaction between the effects of salinity and light intensity for all carotenoids, except for zeaxanthin. The highest increases in most carotenoids were observed in the presence of marine salts with increasing light intensity.

Figure 3 presents the results as a percentage of biomass (dry weight, DW), separated into panels A, B, and C for LL, ML, and HL exposure, respectively. The statistical analyses in Figure 3 refer to the results obtained after growth at each light intensity.

In particular, under LL, the synechoxanthin content in the cultures grown in the presence of 20 g L⁻¹ and 40 g L⁻¹ marine salts increased by 1.6- and 2.0-fold compared to that in the control cultures, respectively. The echinenone and β-carotene contents were also highest at the end of growth with 20 g L⁻¹ and 40 g L⁻¹ marine salts: the echinenone content was 2.7- and 1.9-fold and the β-carotene content was 2.9- and 2.3-fold higher than that of the control cultures, respectively. For all these carotenoids, except for echinenone, significant differences were not detected between the two marine salt concentrations. The contents of myxoxanthophyll and zeaxanthin did not significantly differ among the cultures.

After growth under ML, only myxoxanthophyll did not show significant differences among the cultures. For all other carotenoids, significant differences were detected, with the highest values found in the marine salt treatments.

In both the 20 g L⁻¹ and 40 g L⁻¹ cultures, the highest increase was observed in the echinenone content, which was 3.6-fold higher than that in the control, followed by synechoxanthin (2-fold higher) and β-carotene (2-fold and 1.3-fold higher in 40 g L⁻¹ and 20 g L⁻¹, respectively). In both the 20 g L⁻¹ and 40 g L⁻¹ cultures, zeaxanthin was 1.3-fold higher than in the BG11 cultures.

Under HL, all carotenoids were 1.5- to 2.0-fold higher after growth in marine salt than after growth in BG11, except for synechoxanthin, which did not show significant differences among the cultures.

To further investigate the synergistic effect of these two stress factors, the statistically significant results in all datasets (each carotenoid at all marine salt concentrations and all light intensities) were examined, and they revealed consistent separation between treatment groups according to stress intensity. The results are reported in

Table 3. Carotenoid composition of Synechocystis sp. PCC6803 grown in BG11 and in BG11 + 20 g L⁻¹ and BG11 + 40 g L⁻¹ marine salt at the end of growth (96 h) under 100, 200, and 400 μmoles photons m⁻² s⁻¹. Values are given as % of dry weight (DW) ± standard deviation. For each carotenoid (column), different letters indicate significant differences among the values under all conditions, including different marine salt concentrations and light intensities (p < 0.05).

Light Intensity	Marine Salt Concentration	Synechoxanthin	Myxoxanthophyll	Zeaxanthin	Echinenone	β-Carotene
μmoles Photons m−2 s−1	g L−1	% on DW
100	0	0.022 ± 0.004 a	0.184 ± 0.038 a	0.126 ± 0.018 a	0.030 ± 0.005 a	0.111 ± 0.016 ad
	20	0.046 ± 0.007 ad	0.208 ± 0.043 ad	0.146 ± 0.021 a	0.094 ± 0.014 bd	0.322 ± 0.047 b
	40	0.036 ± 0.006 ad	0.191 ± 0.039 abd	0.134 ± 0.020 a	0.065 ± 0.010 abd	0.256 ± 0.038 b
200	0	0.048 ± 0.008 ad	0.243 ± 0.010 ade	0.135 ± 0.005 a	0.055 ± 0.008 ad	0.077 ± 0.011 a
	20	0.102 ± 0.016 c	0.263 ± 0.004 ade	0.185 ± 0.017 a	0.208 ± 0.031 c	0.107 ± 0.016 ad
	40	0.102 ± 0.016 c	0.242 ± 0.007 abde	0.185 ± 0.018 a	0.198 ± 0.029 c	0.147 ± 0.021 acd
400	0	0.054 ± 0.009 bd	0.173 ± 0.036 a	0.188 ± 0.028 a	0.113 ± 0.011 bde	0.101 ± 0.015 ad
	20	0.053 ± 0.008 bd	0.301 ± 0.062 bde	0.270 ± 0.040 b	0.170 ± 0.025 ce	0.171 ± 0.025 cd
	40	0.063 ± 0.010 bd	0.331 ± 0.068 ce	0.284 ± 0.042 b	0.191 ± 0.028 c	0.148 ± 0.009 acd

.

For synechoxanthin, minimal effects were observed under LL, while a moderate but consistent increase was detected under HL, and the strongest effect was observed at the 200 level, particularly in the 20 g L⁻¹ and 40 g L⁻¹ cultures.

The myxoxanthophyll values exhibited a complex pattern, with multiple mixed-letter groups. Mainly, none of the values in the BG11 cultures or any of the LL cultures showed significant differences. Significant differences emerged in the cultures grown in the highest marine salt concentration under ML and in both the 20 g L⁻¹ and 40 g L⁻¹ cultures under HL, which also showed statistically distinct values from the control cultures.

The zeaxanthin content showed the simplest and clearest results. In all cultures, except for those grown in 20 g L⁻¹ and 40 g L⁻¹ under HL, the values did not show significant differences, demonstrating that the zeaxanthin content was significantly high only under extreme treatment conditions.

The echinenone content showed one of the strongest stratifications among the different conditions. The echinenone content did not differ between any of the cultures grown under LL and the culture grown in BG11 under ML. However, under the most extreme conditions (considering both salt and light stress), higher contents were found after growth in marine salt under ML and HL, and these values were significantly different from those in the LL cultures and the BG11 culture grown under ML.

The β-carotene data showed a moderate degree of differentiation among the treatments. The contents after growth under LL and ML in 20 g L⁻¹ did not significantly differ. In contrast, in the higher-intensity treatments with the highest salt and light stress (particularly 20 g L⁻¹ and 40 g L⁻¹ under HL), the lowest β-carotene content was observed, along with distinct significance groups.

To provide a more detailed view of the results, for each carotenoid, a response surface plot is shown in Figure S1.

Overall, these results indicate clear pigment-specific, marine salt- and light intensity-dependent response patterns. In particular, the results show that myxoxanthophyll and zeaxanthin increased with the increase in both factors, while the synechoxanthin, echinenone, and β-carotene contents were the highest under LL or ML, with no further increase or significant changes when switching from ML to HL. This indicates a loss of synergy with salt stress under HL for the increase in these carotenoids under the tested conditions.

For each light intensity, the chromatograms from the HPLC analysis of the carotenoid extracts are shown in Figures S2, S3, and S4 for LL, ML, and HL, respectively.

2.4. Changes in Photosynthetic Activity

Measurements of the quantum yield of PSII and the oxygen evolution rate showed that, during adaptation to marine salt, the quantum yield of PSII and the oxygen evolution rate of these cultures did not differ from those of the control culture in BG11. Hence, it can be assumed that, during this period, under 50 μmoles photons m⁻² s⁻¹, the cultures with marine salt did not experience stress.

2.4.1. Chlorophyll Fluorescence Changes

Changes in the PSII quantum yield, F_v/F_m, were monitored in the culture samples during growth under different light intensities, namely, 100 (LL) and 400 (HL) μmoles photons m⁻² s⁻¹, and the results are shown in Figure 4, panels A and B, respectively. At the beginning of exposure to LL, the F_v/F_m values did not significantly differ in any of the cultures. During growth at 100 μmoles photons m⁻² s⁻¹, the F_v/F_m values increased in all cultures, and, after 96 h, the increase was higher in BG11 than in the 20 gL⁻¹ and 40 L⁻¹ cultures (by 20%, 12%, and 7%, respectively). However, at the end of growth, none of the cultures showed significant differences in F_v/F_m, indicating that the maximum efficiency of the photosynthetic apparatus was not affected by salinity stress (Figure 4A).

As observed under LL, at 400 μmoles photons m⁻² s⁻¹, the F_v/F_m values did not show significant differences in any of the cultures (Figure 4B). After an initial decrease within the first 24 h, the F_v/F_m values recovered during growth, with the BG11 cultures exhibiting the highest value compared to the 20 g L⁻¹ and 40 g L⁻¹ cultures. Despite this recovery, the final F_v/F_m values were lower than the initial ones. Interestingly, the smallest decline was detected in the BG11 cultures (4%), whereas with marine salt, the decline was the greatest (21% and 15% for 20 g L⁻¹, and 40 g L⁻¹, respectively). However, at the end of growth, as observed at 100 μmoles photons m⁻² s⁻¹, none of the cultures exhibited significant differences in F_v/F_m, indicating that the PSII maximum quantum yield was not affected by salinity stress (Figure 4B) and that, under these conditions, the cultures were fully acclimated to salt and high light. It should be noted, however, that at the lowest and highest light intensities, the F_v/F_m values averaged 0.543 and 0.395, respectively (with the latter being lower by 37%), indicating that the highest light intensity caused a reduction in photosynthetic activity. The changes in non-photochemical quenching (NPQ) under LL and HL with the different marine salt concentrations are reported in Figure 5A and Figure 5B, respectively.

The results showed that, under LL, the presence of marine salt promoted the highest increases in NPQ, with the highest values observed in the 40 g L⁻¹ culture (showing a four-fold increase in NPQ values compared to the BG11 and 20 g L⁻¹ cultures). No significant differences were observed in NPQ between the cultures grown in BG11 and those grown in 20 g L⁻¹ marine salt (Figure 5A). Under HL, the cultures with both marine salt concentrations exhibited double the NPQ value compared to the BG11 cultures (Figure 5B).

2.4.2. Oxygen Evolution and Uptake Measurements

The results of the measurement of oxygen evolution and uptake in cultures grown under LL and HL are reported in Figure 6, panels A and B, respectively. The results show that, at the lowest light intensity of 100 μmoles photons m⁻² s⁻¹, an increase in the O₂ evolution rate, compared to the initial value, was detected in all cultures. Specifically, increases of 15%, 29%, and 52% in the BG11, 20 g L⁻¹, and 40 g L⁻¹ cultures, respectively, were observed at the end of the growth period (Figure 6A). Although these differences among the final values were not significant, it is noteworthy that the increase correlated with the rising marine salt concentration.

At 400 μmoles photons m⁻² s⁻¹, initially, no strong differences in the O₂ evolution rate were detected (Figure 6B). However, during the subsequent growth period, the increase in the O₂ evolution rate in these cultures was greater than that in all cultures exposed to 100 μmoles photons m⁻² s⁻¹, with increments of 65%, 54%, and 41% in the BG11, 20 g L⁻¹, and 40 g L⁻¹ cultures, respectively, at the end of the growth period. As observed at 100 μmoles photons m⁻² s⁻¹, the highest values were maintained in the cultures with marine salt throughout the growth period. However, under HL, after 96 h, the cultures with the highest salt concentration showed an O₂ evolution rate 20% lower than that of the other two cultures.

Regarding the O₂ uptake values, it is noteworthy that, at both light intensities, the respiration rate increased during growth and with increasing salinity (Figure 6A,B). At 100 μmoles photons m⁻² s⁻¹, the cultures grown in 20 g L⁻¹ and 40 g L⁻¹ marine salt reached values 1.9-fold and 2.5-fold higher than those of the BG11 cultures at the end of growth, respectively. At 400 μmoles photons m⁻² s⁻¹, the differences were less pronounced, whereas strong differences were observed between the highest salinity concentration and the BG11 culture (values found in 20 and 40 g L⁻¹ were 1.7-fold and 3.1-fold higher than those found in BG11, respectively).

2.5. Changes in the Biochemical Composition of Synechocystis Biomass

The results for the total carbohydrate and protein contents at the end of growth under LL and HL are reported in Figure 7. The largest differences were induced by salinity stress under the highest light intensity. At 100 μmoles photons m⁻² s⁻¹, the biomass composition showed that marine salt led to a significant increase in carbohydrates compared to the control (BG11) (Figure 7A). In cultures grown with 20 and 40 g L ⁻¹ marine salt, the carbohydrate content increased by 44% and 50%, respectively, compared to that in the control. In contrast, at 400 μmoles photons m⁻² s⁻¹, the carbohydrate content decreased with increasing salinity, reaching the lowest value, being 49% and 57% lower than in the BG11 cultures with 20 and 40 g L⁻¹ marine salt, respectively (Figure 7B). Additionally, the results showed that, in BG11, the carbohydrate content at the end of growth was the same under both light intensities. However, with both marine salt concentrations, at 400 μmoles photons m⁻² s⁻¹, the carbohydrate content was on average 70% lower than at 100 μmoles photons m⁻² s⁻¹.

At the lowest light intensity, the protein content at the end of growth did not significantly differ in any of the cultures (Figure 7C). In contrast, at the highest light intensity, the highest protein content was found in the cultures with both marine salt concentrations (at least 70% higher than in the BG11 culture) (Figure 7D). For the protein content, large differences were observed between the values found in the BG11 cultures, with the value at 400 μmoles photons m⁻² s⁻¹ being 42% lower than that at 100 μmoles photons m⁻² s⁻¹ (Figure 7C,D). Large differences were not observed with either of the marine salt concentrations.

3. Discussion

The results reported here demonstrate the ability of Synechocystis to grow in marine salt at 20 and 40 g L⁻¹, that is, at a salinity higher than that of seawater (35 g L⁻¹). Salinity higher than that typically found in seawater can occur when cultures are grown continuously, with evaporation compensated for by using seawater instead of deionised water. This is important from an eco-friendly perspective. The freshwater required for the cultivation of Synechocystis would be reduced by using sea or salt water, which would have a significant positive impact on large-scale outdoor cultivation. Moreover, high salinity can act as a barrier against competing organisms, making it possible to cultivate Synechocystis in less expensive culture systems such as open ponds.

Due to its susceptibility to contamination, Synechocystis is preferably grown in closed photobioreactors, which reduce but do not eliminate contamination when using BG11 in fresh water. At a commercial scale, Synechocystis cultures remain susceptible to pest infections, even in closed systems, because of insufficient sterilisation of culture equipment, supplemented air, and large volumes of water. To reduce contamination from freshwater grazers and the use of freshwater, and to enable the cultivation of Synechocystis in open ponds, the cultivation of this cyanobacterium in seawater represents a useful approach [30,31]. Concerning growth, the results show that the cyanobacterium was able to grow at different light intensities and salt concentrations, although with some significant differences under LL. These findings are consistent with the data reported by the authors of [32], who used the same Synechocystis strain and found no significant difference in growth at a salinity of 30 g L⁻¹ compared to the control.

The presence of microelements in marine salt, such as iron (Fe), manganese (Mn), zinc (Zn), calcium (Ca²⁺), and chloride (Cl⁻), plays a crucial role in the efficiency of the photosynthetic apparatus. It has been observed that increasing the concentrations of Fe, Mn, and Zn has a positive effect on the growth rate of and biomass accumulation in microalgae [33]. Although BG11 contains cations and anions, the addition of 20 g L⁻¹ and 40 g L⁻¹ marine salt introduces additional microelements.

Compared to the control cultures, salt stress was more pronounced under LL than under HL, probably because the cultures exposed to HL were better adapted to the presence of marine salt due to their longer exposure to this medium. Salt acclimation responses in Synechocystis sp. PCC 6803 include the upregulation of protective carotenoid pigments such as zeaxanthin and myxoxanthophyll under high-salt conditions [34]. This suggests that carotenoid accumulation is a key response to salinity stress, aiding the reactivation of photosynthesis and translation during salt acclimation. Our results support this assumption, as most carotenoids increased in cells grown with marine salt, showing that carotenoid accumulation is strongly dependent on salinity and light intensity stress and that each pigment exhibits a distinct physiological threshold in its responsiveness.

Light intensity was the parameter that consistently drove the strongest and most significant increases in carotenoid accumulation (especially from 100 to 400 µmoles m⁻² s⁻¹). Salt concentration also contributed, but its effect was secondary, often enhancing or modulating the light-driven response rather than acting as the main trigger. All the carotenoids, except for synechoxanthin and β-carotene, increased after growth at the highest light intensities.

The lowest β-carotene content observed in the HL cultures with marine salt could be explained by its preferential contribution to the synthesis of echinenone and zeaxanthin, along with the sustained pathway for myxoxanthophyll synthesis. By contrast, the promotion of this pathway in the LL and ML cultures with marine salt was lower than that in the HL cultures, as revealed by the highest amount of β-carotene, and the lowest amount of myxoxanthophyll s [35,36,37,38].

In our experiments, under LL, the effect of salinity stress on PSII performance was not significant, as shown by the O₂ evolution rate, which even increased with marine salt during growth. In these cultures, acclimation to marine salt influenced the carotenoid composition at the beginning of the LL exposure, although at a relatively low light intensity (50 µmoles photons m⁻² s⁻¹). Indeed, the echinenone and β carotene contents in the 20 and 40 g L⁻¹ cultures were higher than in the BG11 cultures, suggesting that they played a role in protecting the photosynthetic apparatus during growth at 100 µmoles photons m⁻² s⁻¹.

These results show how Synechocystis was able to acclimate to salt stress and maintain efficient PSII activity under LL in the presence of marine salt. Our results also align with previous findings in the Synechocystis sp. CCNM 2501, which showed a marked increase in the carotenoid content under high-salinity stress. In this strain, β-carotene levels were found to be over three times higher under 1 M salinity than under non-saline conditions. Similarly, echinenone levels increased more than four-fold in specific nutrient media under salt stress [12]. On the other hand, at the beginning of the HL exposure, the photosynthetic performance, assessed via F_v/F_m and the O₂ evolution rate, was not much lower or was even the same as that found in the LL cultures, both those grown with BG11 and marine salt. However, the BG11 cultures maintained a lower maximum PSII quantum yield than the marine salt cultures for the first 48 h; thereafter, F_v/F_m tended towards a common value at the end of the growth period. The photosynthetic activity was not strongly affected, as PSII photoprotection was promoted by the presence of carotenoids at the beginning of the exposure, which dissipated excess energy. The synechoxanthin, zeaxanthin, echinenone, and β-carotene contents increased in the cultures during growth under ML. However, due to the effect of light acclimation, these cultures were able to cope with HL exposure. The photosynthetic activity did not show a strong decrease, demonstrating the effect of carotenoids in sustaining the mechanism of PSII repair and protection.

The involvement of carotenoids in the response to high-light stress in Synechocystis has already been reported, as indicated by increased carotenoid synthesis. Carotenoids function as both light-harvesting molecules and potent antioxidants, counteracting the effects of increased ROS production, which can damage cellular components. The induction of de novo biosynthesis was previously detected when cells were exposed to high light intensities (e.g., 500 µmoles m⁻² s⁻¹) [39]. Under these conditions, an increased biosynthesis of carotenoids such as myxoxanthophyll, echinenone, and β-carotene was observed, helping to quench singlet oxygen and dissipate excess energy. Myxoxanthophyll provides the strongest protective effect against oxidative damage, while echinenone is notable for its stability under photo-oxidative stress. More recent studies have reported significantly increased carotenoid accumulation in Synechocystis sp. PCC 6803, achieved by overexpressing carotenoid biosynthetic genes [40]. All modified strains showed an increased accumulation of zeaxanthin and echinenone while maintaining a sizable amount of myxoxanthophyll.

Previous studies on Synechocystis sp. PCC6803 cells have demonstrated their resistance to high-light stress, entering the photoinhibition state only at 800 µmoles photons m⁻² s⁻¹ [41]. However, the effect of simultaneous exposure of Synechocystis cells to varying light intensities and marine salt concentrations has not yet been thoroughly investigated. Cells exposed to light intensity above their saturation irradiance, combined with salt stress, adopt an efficient strategy to reduce PSII overexcitation by dissipating excess energy via NPQ. In our experiments, increasing light intensity and marine salt concentration resulted in the highest NPQ values, which corresponded to the greatest increase in carotenoids, namely, the xanthophylls myxoxanthophyll, zeaxanthin, and echinenone, known to act as strong antioxidants, quenching PQ pool overreduction due to excess energy [4,5,6]. In particular, the ketocarotenoid echinenone is a component of the orange carotenoid protein (OCP), which is essential for triggering photoprotective mechanisms in cyanobacteria [42]. This protein decreases the excess absorbed energy reaching the photosynthetic reaction centres by increasing thermal dissipation at the level of the phycobilisomes, the cyanobacterial light-harvesting antennae [42]. This mechanism is entirely different from that in microalgae and higher plants, which is based on the well-known xanthophyll cycle involving the conversion of violaxanthin to zeaxanthin under high light [4,5].

The strong increase in echinenone, associated with increased light exposure and rising salinity, is consistent with the role of this carotenoid in protecting Synechocystis from high-stress conditions. In our experiments, Synechocystis sp. PCC6803 showed a remarkable capacity to acclimate to high-light and -salinity stress, as indicated by stable F_v/F_m values and increased growth and photosynthetic activity with rising irradiance. Similar results have been reported for Synechocystis sp. PCC 6803 exposed to different light intensities (200 and 1000 μmoles photons m⁻² s⁻¹) and then to strong light at 2000 μmoles photons m⁻² s⁻¹ [37]. In these experiments, a mutant strain lacking zeaxanthin, echinenone, and myxoxanthophyll showed greater PSII impairment than the wild type, indicating that high levels of carotenoids are necessary to sustain protein synthesis and PSII protection under high light. These findings are consistent with our results, which showed that prior adaptation to different light intensities effectively induced increases in echinenone and zeaxanthin, and, more importantly, the presence of marine salt further enhanced the accumulation of these carotenoids.

When examining the O₂ uptake values, a noteworthy finding emerged: during growth, the respiration rate increased at all light intensities in all cultures, and O₂ consumption increased particularly with salinity, reaching values up to twice as high in the culture with 40 g L⁻¹ marine salt than in that with BG11. These findings are consistent with those of previous studies on Synechocystis sp. PCC6803 salt-stressed cultures, which revealed that inhibition of photosynthetic activity due to salinity was associated with an increase in the respiratory electron transport chain, a strategy used to decrease the over-reduction of the photosynthetic apparatus [11,43,44,45]. Increased respiration due to rising salinity is a common phenomenon in microalgae and cyanobacteria, which may indicate a high metabolic cost of salt acclimation (e.g., ion pumping and the synthesis of compatible solutes), thereby diverting energy and carbon from growth and potentially influencing carotenoid precursor availability. These results are of great importance considering the use of this strain for cultivation under outdoor stress conditions. This strain has attracted attention due to the ease of its genetic manipulation and has recently gained importance due to its potential use in various sectors of biotechnology, such as H₂ production [31]. The hydrogenase enzyme is sensitive to the presence of oxygen, and a high O₂ respiration rate could help to avoid or reduce the inhibition of H₂ biosynthesis [46].

This study also examined the growth and biochemical composition of Synechocystis biomass under varying light intensities and salinity levels. The results show that biomass accumulation remained consistent across different salinity conditions, in agreement with [32], in which, for the Synechocystis strain, no significant difference in growth was detected at a salinity of 30 g L⁻¹ compared to the control [32]. Regarding the chlorophyll-a concentration, salinity affected the growth of Synechocystis at the lowest light intensity, as chlorophyll accumulation was lowest in the presence of marine salt, in accordance with previous findings [32]. However, at the highest light intensity, the differences among the different salinities were not significant, probably as a consequence of prior adaptation to various salinities and specific light intensities. Furthermore, in our experiments, the previous adaptation of the HL cultures to ML (200 μmoles photons m⁻² s⁻¹) and to marine salt helped the HL cultures to achieve a higher biomass accumulation than the LL cultures Hence, all the HL cultures were denser than the LL cultures at the end of growth. This may explain why, in the HL cultures, the carotenoid/chlorophyll ratio was lower than in the LL cultures at the end of growth. These findings indicate that the effect of light was more pronounced than that of salt stress in the HL cultures. For this reason, further increases in light intensity would not be effective in studying the synergy between light intensity and salinity stress.

The carbohydrate content increased under salt stress at a moderate light intensity (100 μmoles photons m⁻² s⁻¹) but decreased at a higher light intensity (400 μmoles photons m⁻² s⁻¹), likely due to light limitation at the cellular level as a consequence of the highest biomass. The accumulation of carbohydrates under stress conditions in cyanobacteria is a common strategy used to temporarily store excess reducing power that cannot be utilised for growth. The excess carbohydrates synthesised during the light period are usually utilised at night to sustain protein synthesis. This behaviour has been well documented in outdoor Arthrospira cultures exposed to high-light and -temperature stress [47]. In our experiments, under LL exposure, both chlorophyll and biomass (dry weight) accumulation were lower than those observed under HL exposure at the end of the growth period. These findings may support the idea that LL cultures direct metabolism towards carbohydrate synthesis when growth is reduced. This is also consistent with the different levels of chlorophyll increase observed in the absence and presence of marine salt. The protein content remained stable across conditions, with a slight increase at high light and salinity. In particular, under HL, due to the previous adaptation to marine salt and ML, the HL cultures were able to react to HL exposure by producing higher amounts of carotenoids than the LL cultures; this was more pronounced in the presence of marine salt, which may have exerted greater stress. These findings show that the BG11 cultures activated physiological responses to high-light stress at a lower level than those with marine salt. As a result, the BG11 cultures showed reduced protein synthesis as an effect of the stress and shifted metabolism towards carbohydrate accumulation, associated with biomass accumulation, as reflected by them exhibiting the highest increase in dry weight when compared to the marine salt cultures. In addition, the HL cultures had previously been exposed to ML (200 µmol m⁻² s⁻¹), and the marine salt cultures under ML showed the highest contents of most carotenoids; hence, these cultures were more active in responding to HL stress, corresponding to the maintenance of a higher protein level.

The biochemical composition (carbohydrates ~18% and proteins ~64%) is consistent with that in previous studies [48] and indicates that the cultures were healthy and suitable for biorefinery applications.

Carotenoids play an important role in optimising and maintaining the efficiency of the photosynthetic apparatus due to their functions as light harvesters and antioxidants. Under stress conditions, their increase is related to their role in quenching the over-reduction of the electron transport chain, mainly at the plastoquinone (PQ) pool of the photosynthetic apparatus, which occurs under stress due to excess light energy or low efficiency in driving electrons from PSII to PSI [7,8,14]. The occurrence of PQ pool over-reduction for any of these reasons promotes the formation and accumulation of ROS, which may cause serious damage to the photosynthetic apparatus. This has been well documented in the outdoor cultivation of photosynthetic microorganisms, where cells are exposed to high light intensities [46]. For this reason, the findings regarding the ability to adapt Synechocystis cells to increasing light intensities are useful for setting up inocula of this cyanobacterium for outdoor cultivation. Moreover, biotechnologically, it is well known that outdoor cultures of Synechocystis are subject to heavy grazing by the golden microalga Poterioochromonas malhamensis, which may result in the loss of the culture within a few days of its detection. Indeed, this Chrysophyta actively grazes on Synechocystis cells both during the day and at night, and it can therefore rapidly outgrow the much slower photosynthetic growth of Synechocystis cells. Some strategies have been proposed to control this organism, such as increasing pH to 11 [29] and using phosphite, which provides a competitive growth advantage against microbial contaminants that compete for phosphate sources [49]. However, increasing the salinity of the culture medium is another possible approach, facilitating its cultivation in mass outdoor cultures and improving economic viability.

4. Materials and Methods

The design of the experiment is shown in Figure S5. According to the DOE, cultures adapted to 20 g L⁻¹ and 40 g L⁻¹ marine salt were exposed to increasing light intensities to study the synergistic effects of salt and light stress. The DOE is based on the literature referenced in this manuscript.

4.1. Growth and Culture Conditions

Synechocystis sp. PCC6803 inoculum was grown in BG11 medium [50] under artificial irradiance of 50 μmoles photons m⁻² s⁻¹, provided from one side, in bubbled column photobioreactors (i.d = 50 mm, 400 mL working volume) at 28 °C, and bubbled with an air-CO₂ mixture (97/3 v/v) at a continuous flow rate of 5 dm³ min⁻¹. To evaluate growth in salt medium, different concentrations of marine salt Tropic Marin Zoo Mix (Tropic Marin^® SEA SALT, Wartenberg, Germany) were used by adding 20 and 40 g L⁻¹ marine salt to the BG11 medium; BG11 medium alone served as the control. These salt concentrations were chosen because 35 g L⁻¹ is the salinity of seawater; lower salinities were not used to prevent contamination and save fresh water, and salinities higher than 40 g L⁻¹ were not tested, as this concentration already exceeds seawater salinity and could be useful for assessing the effects of increased salinity due to evaporation.

To adapt Synechocystis cells to these salinity concentrations, they were transferred to BG11 with 20 and 40 g L⁻¹ marine salt and cultivated in these media for at least one month in a bubbled column photobioreactor at 50 μmoles photons m⁻² s⁻¹, under the same conditions as the inoculum cultivation, as previously described. The results on the effect of salt stress on light adaptation were obtained by considering light intensities of 100, 200, and 400 μmoles photons m⁻² s⁻¹, corresponding to LL, ML, and HL, respectively, at an initial dry biomass concentration of 0.3–0.5 g L⁻¹. Further adaptation to increasing light intensity was not considered because the results indicated that the synergistic effect with the marine salt concentrations used was lost under HL.

For this purpose, the cultures in BG11 and those adapted with marine salt, initially maintained at 50 μmoles photons m⁻² s⁻¹, were exposed to 100 μmoles photons m⁻² s⁻¹ to monitor growth and physiological responses to this light intensity. Before exposure to 400 μmoles photons m⁻² s⁻¹, the light intensity was gradually increased, with a one-week period at each intensity: first to 100, then to 200, and finally to 400 μmol photons m⁻² s⁻¹. At all considered light intensities, LL and HL, exposure lasted until the stationary phase was reached. A volume of 0.800 L of the cultures in BG11 and with marine salt (20 and 40 g L⁻¹; hereafter referred to as BG11, 20 g L⁻¹, and 40 g L⁻¹ cultures) was placed in Roux bottles (1 L working volume) and then exposed to 100, 200, and 400 μmoles photons m⁻² s⁻¹, supplied from one side. A 12 h/12 h light/dark cycle was applied. The culture temperature was maintained at 28 °C and bubbled with an air-CO₂ mixture (97/3 v/v) at a continuous flow rate of 5 dm³ min⁻¹. Samples for analysis of biomass composition and monitoring of photosynthetic activity were collected after 8 h of light exposure. This time point was chosen because it was determined to be sufficient to induce photoprotective responses. In this case, the increase in NPQ demonstrated that 8 h of exposure at the different light intensities was sufficient. All experiments were carried out in triplicate. The irradiance at the culture surface was measured using a quantum/radiometer/photometer (LI-250A, Li-Cor Biosciences, Lincoln, NE, USA) equipped with a quantum cosine-corrected sensor.

The pH of the initial medium was 7.3 ± 0.1. During the experiment, the pH was monitored at the time of sample collection for measurements. The pH value remained constant during the growth period.

4.2. Chlorophyll, Carotenoids, and Dry Weight Determination

The chlorophyll and total carotenoid concentrations were determined spectrophotometrically in triplicate samples. Five millilitres of culture was centrifuged in glass tubes for 8 min at 2650× g in an ALCPK110 centrifuge. The supernatant was discarded, and the pellet was resuspended in 5 mL of pure methanol. The tubes were then placed in a 70 °C water bath for 3 min and centrifuged again for 8 min at 2650× g. The absorbance of the supernatant was measured at 665 and 750 nm against pure methanol and calculated according to [51]:

Chl a (mg L⁻¹) = 12.61 × (Abs666 − Abs750)

Carot. Tot (mg L⁻¹) = 4.08 × (Abs470 − Abs750) − 0.0117 × Chl a

In the same extracts, the concentration of individual carotenoids was determined by reversed-phase HPLC using a Beckman System Gold (module 125 solvent) with a diode-array detector (DAD), model 168 Nouveau, and a Luna C8 column (Phenomenex, Torrance, CA, USA), according to [52]. All reagents were HPLC-grade, except for the tetrabutyl ammonium acetate salt solution, pH 6.5, which was prepared with distilled water and filtered. Eluent solutions were solvent A, 80:20 (v/v) methanol, 28 mM tetrabutyl ammonium acetate; solvent B, 100% methanol. The flow rate for elution was 0.8 mL/min. Throughout the analysis, a linear gradient was used. The elution protocol was as follows: start with 100% solvent A, reach 100% solvent B in 25 min, maintain for 2 min, then return to 100% solvent A in 3 min.

Carotenoids were identified by comparing the retention times and spectra with HPLC-grade standards (Sigma-Aldrich, Louis, MO, USA). Quantification was obtained using calibration curves made with the respective standards. The analysis was performed in triplicate.

The dry weight was determined by filtering the samples onto a glass fibre membrane (Whatman GF/F, Maidstone, UK) that had a 47 mm diameter and 1.2 μm porosity and was previously dried in an oven at 105 °C. The filters were then washed with 50 mL of distilled water to remove residual salts and kept for 2 h in an oven at 105 °C before weighing.

A Response Surface Model (RSM) was employed to assess both the individual and combined effects of the independent variables on the selected dependent variables, which were chosen as response factors. The interaction between pairs of independent variables and their impact on the responses are illustrated using 3D surface plots. Implementation was done in Python 3.12.

4.3. Fluorescence Measurements

The fluorescence of chlorophyll-a was measured using two pulse-amplitude modulation fluorometers (PAM-2000, H. Walz, Effeltrich, Germany). The nomenclature of fluorescence follows [53]. The minimum fluorescence, F₀, was measured with modulated light (<0.3 µmoles m⁻² s⁻¹) from a light-emitting diode (peak wavelength at 655 nm, 600 Hz). In the dark, cyanobacteria are usually found in state II, with lower fluorescence, yielding an F_m value lower than F′_m. Therefore, the true F_m value was measured under red light illumination (150 µmoles photons m⁻² s⁻¹) in the presence of 1 × 10⁻⁵ M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which prevents induction of electron transport between the primary quinone electron accepter (Q_A) and secondary quinone electron accepter (Q_B) on the reducing side of PSII. The true F_m was used for calculation of the F_v/F_m ratio and NPQ [54].

4.4. Oxygen Measurements

A biological oxygen monitor (Yellow Spring Instrument Co., Inc., Yellow Springs, OH, USA), YSI model 5300, was used to quantify the cultures’ photosynthetic oxygen evolution rates. Measurements of oxygen evolution were conducted at a constant temperature of 28 °C and a saturating irradiance of 850 µmoles photons m⁻² s⁻¹. Before the measurements, samples were taken from the culture containers with minimal turbulence, immediately transferred to the oxygen measurement cuvettes, and flushed with nitrogen. The flushing with nitrogen was done by means of a needle connected to the nitrogen source, bubbling the nitrogen directly into the culture sample placed in the chamber for the oxygen concentration measurement. This procedure ensured that the oxygen already present in the culture sample was lowered so that the oxygen produced during the measurements did not reach saturation inside the sample. Each measurement was repeated three times.

4.5. Biochemical Characterisation of the Biomass

To determine the biochemical composition, biomass samples were collected by centrifugation, washed with deionised water to remove salts, and frozen before analysis. The total carbohydrate content was determined using the phenol–sulphuric acid method according to [55] with D+ glucose used as a standard. The total protein content was determined according to [56]. A calibration curve was prepared with standard solutions of Bovine Serum Albumin (BSA).

4.6. Statistical Analyses

To assess variations in the impact of different marine salt concentrations at each light intensity, a one-way ANOVA was used to analyse the data, and Tukey’s multiple range test was used to identify significant differences (p < 0.05). A two-way ANOVA was used to analyse data collected from cultures exposed to different salinities and light intensities. The PRISM 5 software programme was used for statistical analysis.

5. Conclusions

The experiments evaluated the extent to which Synechocystis sp. PCC6803 can acclimate to and grow under stepwise increases in salt concentration and light intensity, and how biomass concentration, biochemical composition, and physiological parameters change. The findings revealed the potential use of seawater for cultivating the freshwater cyanobacterium Synechocystis sp. PCC6803, which was found to grow in media with different salt concentrations without significant differences to the control medium. Based on the results obtained at 40 g L⁻¹ salinity, it can be stated that natural seawater could also be used to cultivate Synechocystis sp. PCC6803, with the advantage that natural seawater is a free and abundant resource suitable for large-scale bioenergy production. The use of seawater for the cultivation of Synechocystis sp. PCC6803 offers several advantages; for example, it helps to reduce freshwater consumption for large-scale cultivation and protects Synechosystis sp. PCC6803 cultures from grazers that can contaminate them, a phenomenon that can occur especially when managing outdoor large-scale photobioreactors.

Synechocystis cultures showed strong resilience to the combined effects of salt and light stress, which resulted in a moderate reduction in productivity compared to unsalted cultures. However, processing the biomass may result in additional production costs due to the need to wash the biomass after harvesting. On the other hand, even considering the need to wash the biomass, the freshwater savings resulting from industrial-scale Synechocystis production in seawater would still be advantageous in terms of both production costs and environmental impact. Furthermore, in seawater, the higher carotenoid content adds value to the biomass, shifting the cost balance positively. It should also be considered that washing the biomass is not always necessary.

The fact that a well-balanced biochemical composition was maintained in Synechocystis sp. PCC6803 biomass under the different conditions tested indicates that the cultures were healthy and adds value to the leftover biomass for biorefinery purposes after biohydrogen production.

All these aspects represent advantages in terms of the cost-effectiveness and sustainability of the production process (and therefore in terms of antioxidant-enriched biomass of biohydrogen production) and are essential for achieving a promising increase in growth performance in outdoor cultivation in seawater, where the cyanobacterium will be more easily subject to contamination and will also be exposed to light intensities higher than those tested.