Multi-factor effects of culture conditions on lutein and β-carotene production in Iranian Dunaliella sp. isolates

Abstract

Carotenoids, including lutein and β-carotene, are valuable natural pigments with extensive applications in the food, pharmaceutical, and nutraceutical industries. In the current study, using the factorial experimental design, the biomass and carotenoid production in three Iranian Dunaliella sp. isolates (ABRIINW-B8, ABRIINW-G23, ABRIINW-I44) under combinations of light regime, salinity, nitrate, and copper supplementation, and cultivation time were surveyed. Significant strain and treatment-dependent differences were observed in biomass accumulation and pigment production. Within the tested experimental ranges, strain B8 showed the highest carotenoid accumulation under a light regime (1200 µmol photons m⁻² s⁻¹), 1 M NaCl, 0.5 M KNO₃, and 0.05 mM CuSO₄·7 H₂O, reaching 11.5 mg g⁻¹ DW lutein (7.36 mg L⁻¹) and 63.5 mg g⁻¹ DW β-carotene (40.64 mg L⁻¹). Strain G23 exhibited the highest chlorophyll content. These findings demonstrate the capacity of Iranian Dunaliella isolates to produce high-value carotenoids and identify culture conditions that enhance lutein and β-carotene accumulation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08587-7.

Introduction

Microalgae, which are rich in proteins, essential amino acids, vitamins, polyunsaturated fatty acids, and carotenoids, are increasingly recognized as promising nutritional resources for future food and bio-based industries [1–3]. As photosynthetic microorganisms, they exhibit high photosynthetic efficiency and substantial carbon sequestration capacity while requiring considerably less arable land and freshwater than conventional crops, positioning them as sustainable platforms for novel food and nutraceutical products [3–5].

Among microalgae, halophilic species offer particular industrial advantages because they can thrive under extreme salinities and other harsh environmental conditions [6]. Dunaliella spp., halophilic green microalgae, exhibit key physiological traits that enable adaptation to fluctuating salinity and light, making them highly attractive for large-scale cultivation [7, 8]. Optimal growth typically occurs within a specific salinity range; several studies report optimal performance around 0.5–2.0 M NaCl, with growth sustained across a broad salinity spectrum while maintaining low intracellular sodium levels and high photosynthetic efficiency in D. salina [9, 10]. Under such stress conditions, D. salina can redirect metabolic flux toward the synthesis of high-value compounds, particularly carotenoids and lipids, thereby enhancing its biotechnological relevance [9, 11].

D. salina is recognized as one of the richest natural sources of β-carotene, capable of accumulating very high intracellular levels of this pigment under suitable stress conditions [12]. D. salina biomass is widely recognized for its strong antioxidant properties and, based on toxicological studies and regulatory assessments, has been classified as safe for use as a food ingredient and supplement by European authorities [13, 14]. In addition to β-carotene, this microalga can also produce substantial amounts of lutein, which is increasingly exploited in dietary supplements and health products [15]. These carotenoids are extensively employed as natural colorants, nutraceuticals, and functional ingredients in the food, pharmaceutical, cosmetic, and wellness industries, contributing to skin protection, eye health, and the prevention of oxidative stress–related diseases [16–18]. In addition to their use as natural colorants, carotenoids such as β-carotene and lutein function as effective quenchers of singlet oxygen and free radicals, protecting cellular components from oxidative damage and contributing to their antioxidant activity in biological systems [19, 20]. In vitro and in vivo studies have demonstrated that xanthophylls like lutein can exhibit comparable or even superior radical scavenging activity relative to β-carotene, highlighting complementary antioxidant roles of different carotenoid classes in photoprotection [20, 21]. Compared with chemically synthesized pigments, microalgal carotenoids often show enhanced bioactivity and potential health benefits due to their natural origin and complex structural diversity, which align with increasing demand for sustainable, naturally derived bioactive compounds in food and health industries [20, 22].

Despite the high commercial potential of Dunaliella sp., achieving economically viable carotenoid yields remains challenging, particularly under industrially relevant conditions [11, 23]. Enhancement of lutein and β-carotene production relies heavily on stress-based cultivation strategies that modulate light, salinity, and nutrient availability. Previous studies have shown that high light intensity, nitrogen limitation, and micronutrient stress can markedly increase carotenoid accumulation [24, 25]. Among micronutrients, copper has been identified as an important regulatory element influencing photosynthetic performance and carotenoid biosynthesis in microalgae, particularly through modulation of plastocyanin function and MEP pathway genes in Dunaliella and related species when applied within a non-toxic range (0.01–0.1 mM) [26–29]. Accordingly, copper represents a relevant environmental factor for fine-tuning pigment production under controlled stress conditions.

Most studies to date have examined the effects of individual or a few environmental factors on laboratory-maintained Dunaliella strains, and there is still limited information on how multiple stressors act in combination, especially in native isolates [30, 31]. Comprehensive evaluation of indigenous Iranian Dunaliella strains (B8, G23, I44) is essential to identify superior genotypes with enhanced carotenoid productivity and stress tolerance for regional industrial bioprocesses. In particular, multivariate interactions among salinity, light regime, nitrate availability, and copper supplementation that regulate carotenoid biosynthesis are still insufficiently characterized [32–35].

In the present study, three native Iranian Dunaliella isolates were investigated under a multifactorial design combining light regime (low: 200 µmol photons m⁻² s⁻¹; high: 1200 µmol photons m⁻² s⁻¹), salinity (low: 1 M NaCl; high: 3 M NaCl), nitrate concentration (low: 0 M; high: 0.5 M KNO₃), and copper supplementation (low: 0 mM; high: 0.05 mM CuSO₄·7 H₂O). The ranges of these factors were selected based on previous Dunaliella studies that defined growth-permissive yet stress-inducing conditions, enabling assessment of their individual and interactive effects on biomass productivity and carotenoid accumulation [9, 36].

Materials and methods

Microalga strains and preparation of basal growth medium

Three local Dunaliella strains (B8, G23, and I44, originally designated ABRIINW-B8, ABRIINW-G23, and ABRIINW-I44) were obtained from the Microalgae Culture Collection of the Northwest and West Branch of the Agricultural Biotechnology Research Institute of Iran (ABRIINW), Tabriz, Iran (38.0965° N, 46.2738° E). These strains were originally isolated from Hormozgan Lake (B8), Gavkhooni Marsh (G23), and Urmia Lake (I44), respectively. Strain identification as Dunaliella sp. was confirmed based on morphological characteristics and molecular analysis of partial 18 S rRNA (~ 800 bp) and ITS (~ 600 bp) sequences. The obtained sequences were compared with reference D. salina sequences available in GenBank using BLASTn analysis, confirming their taxonomic assignment within the genus Dunaliella. The three strains were deposited in the local ABRIINW microalgae bank with accession numbers 51,245, 51,265, and 51,268, respectively.

Isolates were cultivated in a basal medium containing 1 M NaCl, 0.048 M MgCl₂·6 H₂O, 0.00036 M CaCl₂·2 H₂O, 0.0225 M Na₂SO₄, 0.0049 M K₂SO₄, and 0.1 M Tris-base. To 985 mL of autoclaved basal medium, 5 mL each of sterilized stock solutions were added: 0.5 M KNO₃, 0.1 M KH₂PO₄, and a trace element solution containing 0.0168 mM CoCl₂·6 H₂O, 0.0036 mM MnCl₂·4 H₂O, 0.0011 mM Na₂MoO₄·2 H₂O, 0.0136 M Na₂EDTA, 0.0047 M FeCl₃·6 H₂O, and 0.05 mM CuSO₄·7 H₂O, 0.017 mM ZnSO4·7 H₂O. Cultures were maintained at 25 ± 1 °C under a 16:8 h light–dark cycle, ensuring optimal growth for all three strains. The basal medium, including 1 M NaCl, macronutrients, and trace elements, was selected to provide a balanced environment that supports normal cellular growth, maintains photosynthetic efficiency, and minimizes stress, serving as a reliable baseline for evaluating the effects of subsequent treatments such as high light, elevated salinity, or nutrient and copper supplementation [31, 37].

Experimental design and replication

A full factorial experimental design was implemented with the following fixed factors: genotype (B8, G23, I44), light regimes (low light: 200 µmol photons m⁻² s⁻¹, 16:8 h light–dark cycle; high light: 1200 µmol photons m⁻² s⁻¹, continuous illumination), salinity (low: 1 M; high: 3 M NaCl), nitrate supplementation (low: 0 M; high: 0.5 M KNO₃), copper supplementation (low: 0 mM; high: 0.05 mM CuSO₄·7 H₂O), and harvest time (0, 1, 7, and 14 days after inoculation). Adjustable LED panels (Heliospectra LX601C, Sweden) were positioned above the cultures to provide light regimes, and light intensity at the culture surface was measured using a quantum sensor (LI-250 A, LI-COR, USA). Salinity was established by dissolving analytical-grade NaCl in the culture medium and verified with a conductivity meter (SevenCompact S230, Mettler-Toledo, Switzerland). Nitrate and copper were introduced from stock solutions to achieve the specified final concentrations. Sampling for biomass and pigment measurements occurred at 0, 1, 7, and 14 days after inoculation.

Cultures were maintained in 250 mL Erlenmeyer flasks containing 100 mL of culture medium. Continuous orbital shaking at 80 rpm ensured adequate mixing and gas exchange, providing sufficient oxygen transfer for small-scale cultures without external aeration. The two light regimes were applied as described above. Under the high light (1200 µmol photons m⁻² s⁻¹), cultures received continuous illumination (24 h), whereas the low-light (200 µmol photons m⁻² s⁻¹) was applied under a 16:8 h light–dark photoperiod in a phytotron. Temperature was maintained at 25 ± 1 °C throughout the experiment. High light flasks were placed in a water bath connected to a temperature-controlled circulator to prevent heat accumulation, while low-light cultures were kept in a temperature-controlled growth chamber. To minimize evaporation, flasks were covered with sterile cotton plugs, and culture volumes were maintained constant over time.

The selected light regimes were chosen to represent moderate and high irradiance conditions (200 vs. 1200 µmol photons m⁻² s⁻¹) that modulate carotenoid accumulation and photophysiological responses in D. salina [12, 38]. Elevated salinity (1 M vs. 3 M NaCl) was applied to induce osmotic stress, a well-established trigger for secondary carotenoid biosynthesis, particularly β‑carotene, in D. salina [12, 38, 39]. Nitrate supplementation (0 vs. 0.5 M KNO₃) was used to evaluate the effect of nitrogen availability on growth and pigment accumulation, while copper supplementation (0 vs. 0.05 mM CuSO₄·7 H₂O) was applied based on the dual role of Cu²⁺ as an essential micronutrient and a potential stressor affecting photosynthetic pigments and oxidative balance [26].

These ranges were selected based on previous studies on Dunaliella sp. and preliminary trials with the indigenous Iranian strains, ensuring that they influenced biomass and carotenoid production without causing severe stress or mortality [9, 37]. Cultures grown under basal conditions (1 M NaCl, low light, with nitrate and copper supplementation) served as control treatments, providing baseline physiological and biochemical data for comparison with stress-induced conditions. Each combination of treatments was replicated three times, and positional effects were minimized by randomizing flask positions in the growth chamber and re-randomizing them daily. Some treatment combinations that resulted in non-viable cultures or unreliable measurements were excluded from the final statistical analysis. The experimental factors and their levels used in the full factorial design are summarized in Table 1.

Table 1.

Experimental factors and levels used for the cultivation and treatment of Dunaliella strains

Experimental factor	Levels/conditions
Genotype	ABRIINW-B8, ABRIINW-G23, ABRIINW-I44
Light regime	Low light: 200 µmol photons m⁻² s⁻¹ (16:8 h light–dark cycle), High light: 1200 µmol photons m⁻² s⁻¹ (continuous illumination, 24 h)
Salinity	1 M NaCl; 3 M NaCl
Nitrate supplementation	0 M KNO₃; 0.5 M KNO₃
Copper supplementation	0 mM CuSO₄·7H₂O; 0.05 mM CuSO₄·7H₂O
Harvest time	0, 1, 7, and 14 days after inoculation
Replication	Three independent biological replicates per treatment

Biomass measurement and optical density monitoring

Growth was monitored by measuring optical density at 730 nm (OD₇₃₀) using a UV–Vis spectrophotometer. Dry biomass was determined as ash-free dry weight (AFDW) following the method described by [40]. For biomass quantification, the following equation was applied:

Quantification of lutein and β-carotene

Pigments were extracted from 10 mL of culture by centrifugation at 5000 rpm for 5 min at room temperature. The resulting pellet was resuspended in 10 mL of analytical-grade acetone and vortexed for 60 s to ensure complete extraction. The suspension was filtered through a 0.2 μm membrane before HPLC analysis, following a method modified from [41]. Pigments were analyzed by HPLC-PDA (detection at 445 nm) on a C18 column using a mobile phase of ethyl acetate: methanol: acetonitrile (30:20:50, v/v/v) at a flow rate of 1.5 mL min⁻¹ and 35 °C, with an injection volume of 20 µL and a total run time of 8 min. Peak integration was performed using Empower 3 software with automatic baseline correction, minimum peak width of 0.04 min, and height threshold > 3× baseline noise. Representative HPLC chromatograms of lutein and β-carotene standards, as well as microalgal extracts, are provided in the Supplementary Information (Supplementary Figs. 1–4). Calibration curves for lutein and β-carotene were prepared from serial dilutions of authentic standards. Peak areas were used to determine the slope, intercept, and coefficient of determination (R²) for each pigment. LOD and LOQ were calculated according to ICH Q2(R1) guidelines [42]. The resulting LOD and LOQ values were 0.2 and 0.62 ppm for lutein, and 0.175 and 0.531 ppm for β-carotene, respectively. Pigment contents were expressed as mg g⁻¹ dry biomass (DW), where DW refers to the ash-free dry weight of algal cells, and as mg L⁻¹ of culture.

Chlorophyll quantification

Chlorophyll a and b were extracted with 100% acetone following the method of [43]. Briefly, 3 mL of culture was centrifuged for 5 min at 5000 rpm; the pellet was resuspended in 3 mL of acetone, vortexed for 30 s, and centrifuged again. Absorbance of the supernatant was measured at 662 and 645 nm in a 3 cm path-length quartz cuvette.

Chlorophyll concentrations in the acetone extracts were calculated using spectrophotometric equations that relate absorbance at 662 and 645 nm to chlorophyll a and b contents, as reported by [44, 45]. Total chlorophyll was obtained as the sum of chlorophyll a and chlorophyll b.

Statistical analysis

Data were analyzed using factorial ANOVA in SAS 9.4, with genotype, light regime, salinity, nitrate, and copper as treatment factors. The experiment included three replicates and was arranged as a split-plot-in-time design, with genotype × light × salinity × nitrate × copper combinations assigned to main plots and harvest time as the subplot factor. Carotenoid (lutein, β-carotene), chlorophyll, and biomass measurements were used as response variables. Homogeneity of variance and normality of residuals were checked for all ANOVAs. Post hoc comparisons were performed using Duncan’s multiple range test (α = 0.05), which allows all pairwise comparisons among treatment means while controlling the Type I error rate. Partial η² values were calculated to quantify each factor’s effect, providing insight into the practical significance of the results. Heat maps of treatment effects were generated using Morpheus software [46].

Results

Effect of environmental factors on biomass accumulation

The effects of genotype, light regime, salinity, nitrate, copper supplementation, and harvest time on biomass accumulation were evaluated in strains B8, G23, and I44. Statistical analysis (Table 2) indicated that genotype and harvest time had significant impacts on biomass production, while the environmental treatments further modulated growth responses. Biomass accumulation patterns were strain-specific, highlighting clear genetic influences on growth performance.

Table 2.

Analysis of variance (ANOVA) for the effects of genotype, environmental treatments, and harvesting time on biomass content in three Dunaliella strains (B8, G23, I44)

Source of variation	df	SS	MS	F	p-value	η2
block	2	0.002	0.001	1.86	p = 0.160	0.0001
a	2	0.022	0.011	14.85	p < 0.001	0.001
block*a	4	0.002	0.000	0.96	p = 0.431	0.0001
b	10	3.355	0.336	453.39	p < 0.001	0.188
a*b	20	0.114	0.006	7.67	p < 0.001	0.006
block*b(a)	60	0.075	0.001	1.68	p = 0.008	0.004
c	3	11.439	3.813	5152.51	p < 0.001	0.641
a*c	6	0.018	0.003	4.06	p < 0.001	0.001
b*c	17	2.630	0.155	209.03	p < 0.001	0.147
a*b*c	34	0.095	0.002	3.79	p < 0.001	0.005
E	120	0.089	0.00074
Total	278	17.841

df  degrees of freedom, SS  Sum of squares, MS  Mean square, η² Effect size

Factor a = genotype (B8, G23, I44), b = environmental treatments, c = harvesting time. Block = experimental replication; E = residual error

Treatment combinations with invalid data were excluded from the analysis data were excluded from the analysis

Across all strains, the high light regime combined with low salinity supported greater biomass production than the low-light regime or high-salinity conditions. High nitrate supplementation markedly enhanced growth, particularly when cultures were harvested on day 14, whereas high copper supplementation had a smaller but positive effect on biomass accumulation. In contrast, low nitrate or low copper conditions constrained growth, often yielding biomass levels comparable to the control, emphasizing the critical role of nutrient availability.

Under the tested high light, low-salinity, high-nitrate, and high-copper conditions, strain B8 achieved the highest biomass, strain B8 achieved the highest biomass, reaching 1.58 ± 0.04 g L⁻¹ on day 14, followed by G23 (1.51 ± 0.05 g L⁻¹) and I44 (1.46 ± 0.03 g L⁻¹). These values represented a substantial increase relative to the corresponding control cultures, in which biomass reached only 0.73 ± 0.02, 0.72 ± 0.03, and 0.71 ± 0.02 g L⁻¹ for B8, G23, and I44, respectively (Fig. 1A-C). Although genotype effects were statistically significant, the absolute differences in maximum biomass among strains remained relatively small, and these trends were consistent across biological replicates (Fig. 2).

Fig. 1.

A-C Biomass accumulation in Dunaliella strains B8, G23, and I44 under different environmental conditions, illustrating the influence of light intensity, salinity, nitrate, copper, and harvesting time. Biomass content (expressed as g L⁻¹) is shown. Treatments are abbreviated HL  High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹), HS  High Salinity (3 M), LS  Low Salinity (1 M), HN  High Nitrate (0.5 M), LN  Low Nitrate (0 M), HC  High Copper (0.05 mM), LC  Low Copper (0 mM). Panels correspond to strains as follows: A, B8; B, G23; C, I44

Fig. 2.

Biomass accumulation in three Dunaliella strains under different growth conditions: light, salinity, nitrate, copper, and harvesting time (0, 1, 7, 14 days). Darker colors represent higher biomass (g L⁻¹). Treatments are abbreviated HL High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹),HS  High Salinity (3 M),LS  Low Salinity (1 M),HN  High Nitrate (0.5 M),LN  Low Nitrate (0 M),HC  High Copper (0.05 mM),LC  Low Copper (0 mM)

Strain-specific responses to environmental stress were observed. G23 maintained relatively stable biomass under the low-light and prolonged cultivation, whereas I44 exhibited reduced growth under high salinity, indicating lower tolerance to severe osmotic stress. Overall, these results demonstrate that biomass production in Dunaliella is strongly influenced by the interplay of genotype, light, salinity, nutrient availability, and harvest timing, with nitrate and copper availability being particularly critical for maximizing growth.

The results showed that the highest volumetric carotenoid productivity (mg L⁻¹) was obtained under high nitrate supplementation (0.5 M KNO₃), which also maximized biomass, rather than under low-nitrate conditions. This indicates that, for these Iranian isolates, efficient photosynthesis and sustained growth under sufficient nitrate support simultaneous biomass formation and carotenoid synthesis, rather than relying solely on stress-induced secondary carotenoid accumulation.

Lutein and β-carotene content under different growth conditions

We evaluated the effects of genotype, light regime, salinity, copper and nitrate supplementation, and harvest time on lutein and β-carotene accumulation in strains B8, G23, and I44. Both genotype and environmental factors significantly influenced carotenoid production, with harvest timing being particularly critical. Maximum carotenoid levels were observed under high light, low salinity, high nitrate, and high copper supplementation, with peak accumulation on day 7. Under these conditions, B8 produced 11.5 mg g⁻¹ DW lutein and 63.5 mg g⁻¹ DW β-carotene, corresponding to volumetric concentrations of 7.36 mg L⁻¹ and 40.64 mg L⁻¹, respectively. G23 accumulated 7.3 mg g⁻¹ DW lutein and 41.5 mg g⁻¹ DW β-carotene, whereas I44 produced 6.5 mg g⁻¹ DW lutein and 51.6 mg g⁻¹ DW β-carotene. In contrast, high-salinity conditions (3 M NaCl) strongly suppressed carotenoid synthesis in all strains. Statistical analysis of lutein and β-carotene data is provided in Table 3 and 4.

Table 3.

Analysis of variance (ANOVA) for the effects of genotype, environmental treatments, and harvesting time on %lutein content in three Dunaliella strains (B8, G23, I44)

Source of variation	df	SS	MS	F	p-value	η2	Partial η2
block	2	1.549	0.774	1.62	P = 0.202	0.0005	0.026
a	2	131.580	65.790	137.26	p < 0.001	0.04	0.696
block*a	4	2.078	0.519	1.08	P = 0.367	0.0006	0.035
b	10	2003.979	200.397	418.10	p < 0.001	0.6070	0.972
a*b	20	69.455	3.472	7.25	p < 0.001	0.021	0.547
block*b(a)	60	22.408	0.373	0.78	P = 0.858	0.006	0.280
c	3	441.857	147.285	307.29	p < 0.001	0.133	0.885
a*c	6	28.669	4.778	9.97	p < 0.001	0.008	0.333
b*c	17	395.112	23.241	48.49	p < 0.001	0.119	0.873
a*b*c	34	148.692	4.373	9.12	p < 0.001	0.45	0.721
E	120	57.516	0.479			0.17
Total	278	3302.901				1.000

df  degrees of freedom, SS Sum of squares, MS Mean square, η² = effect size

Factor a = genotype (B8, G23, I44), b = environmental treatments, c = harvesting time. Block = experimental replication; E = residual error

Treatment combinations with invalid data were excluded from the analysis data were excluded from the analysis

Table 4.

Analysis of variance (ANOVA) for the effects of genotype, environmental treatments, and harvesting time on %β-carotene content in three Dunaliella strains (B8, G23, I44)

Source of variation	df	SS	MS	F	p-value	η2	Partial η2
block	2	4.54	2.27	0.09	p = 0.915	0.0001	0.001
a	2	1019.904	509.952	19.83	p < 0.001	0.038	0.248
block*a	4	89.325	22.331	0.87	p = 0.485	0.003	0.028
b	10	8408.344	840.834	32.70	p < 0.001	0.318	0.731
a*b	20	1801.049	90.052	3.50	p < 0.001	0.068	0.368
block*b(a)	60	1582.571	26.376	1.03	p = 0.444	0.060	0.338
c	3	1784.493	594.831	23.14	p < 0.001	0.067	0.367
a*c	6	870.105	145.017	5.64	p < 0.001	0.033	0.220
b*c	17	5653.467	332.556	12.93	p < 0.001	0.214	0.646
a*b*c	34	2073.186	60.976	2.37	P = 0.0003	0.078	0.402
E	120	3085.248	25.71			0.117
Total	278	26372.236

df degrees of freedom, SS  Sum of squares, MS  Mean square, η² = effect size

Factor a = genotype (B8, G23, I44), b = environmental treatments, c = harvesting time. Block = experimental replication; E = residual error

Treatment combinations with invalid data were excluded from the analysis data were excluded from the analysis

After day 7, lutein and β-carotene levels (mg g⁻¹ DW) declined in all strains, suggesting that extended cultivation under stress limits carotenoid accumulation due to nutrient depletion, pigment degradation, or metabolic reallocation. In B8, lutein decreased from 11.5 mg g⁻¹ DW on day 7 to 8.1 mg g⁻¹ DW on day 14, and β-carotene declined from 63.5 mg g⁻¹ DW to 41.2 mg g⁻¹ DW. Similar trends were observed in G23 and I44. These results highlight clear genotypic differences: B8 consistently had the highest pigment levels, G23 the lowest, and I44 intermediate lutein but relatively low β-carotene. They also indicate that optimal conditions for carotenoid biosynthesis differ from those for maximum biomass production. For example, biomass reached its maximum under high light regime, low salinity, high nitrate, and high copper supplementation at day 14, whereas carotenoid contents peaked at day 7 under the same conditions, illustrating an intrinsic trade-off between biomass formation and carotenoid accumulation in these Dunaliella isolates. Carotenoid accumulation patterns are illustrated in Fig. 3A–C (lutein) and Fig. 4A–C (β-carotene). Heat maps summarizing treatment effects on lutein and β-carotene production are shown in Fig. 5 (lutein) and Fig. 6 (β-carotene).

Fig. 3.

A-C Lutein accumulation in Dunaliella strains B8, G23, and I44 under various environmental conditions, highlighting the effects of light intensity, salinity, nitrate, copper, and harvesting time on biomass production. The lutein content (expressed as mg g⁻¹ DW) is shown. Treatments are abbreviated HL  High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹), HS  High Salinity (3 M), LS  Low Salinity (1 M), HN  High Nitrate (0.5 M), LN  Low Nitrate (0 M), HC  High Copper (0.05 mM), LC  Low Copper (0 mM). Panels correspond to strains as follows: A, B8; B, G23; C, I44

Fig. 4.

A-C β-carotene accumulation in Dunaliella strains B8, G23, and I44 under various environmental conditions, highlighting the effects of light intensity, salinity, nitrate, copper, and harvesting time on biomass production. β-carotene content (expressed as mg g⁻¹ DW) is shown. Treatments are abbreviated HL  High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹), HS High Salinity (3 M), LS  Low Salinity (1 M), HN  High Nitrate (0.5 M), LN  Low Nitrate (0 M), HC  High Copper (0.05 mM), LC  Low Copper (0 mM). Panels correspond to strains as follows: A, B8; B, G23; C, I44

Fig. 5.

Lutein accumulation in three Dunaliella strains under different growth conditions: light, salinity, nitrate, copper, and harvesting time (0, 1, 7, 14 days). Darker colors represent higher lutein content (expressed as mg g⁻¹ DW). Treatments are abbreviated HL  High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹), HS  High Salinity (3 M), LS  Low Salinity (1 M), HN  High Nitrate (0.5 M), LN  Low Nitrate (0 M), HC  High Copper (0.05 mM), LC  Low Copper (0 mM)

Fig. 6.

β-carotene accumulation in three Dunaliella strains under different growth conditions: light, salinity, nitrate, copper, and harvesting time (0, 1, 7, 14 days). Darker colors represent higher β-carotene (mg g⁻¹ DW). Treatments are abbreviated HL  High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹), HS  High Salinity (3 M), LS  Low Salinity (1 M), HN  High Nitrate (0.5 M), LN  Low Nitrate (0 M), HC  High Copper (0.05 mM), LC  Low Copper (0 mM)

Relationship between chlorophyll and carotenoid production in Dunaliella under environmental stress

Figures 7A–C illustrate the primary effects of the applied treatments on chlorophyll and carotenoid production in all Dunaliella strains. An inverse relationship was consistently observed between chlorophyll accumulation and carotenoid synthesis. Conditions that enhanced carotenoid accumulation (high light, low salinity, high nitrate, and high copper supplementation) and shorter cultivation periods were generally associated with decreased chlorophyll levels. Conversely, treatments that promoted chlorophyll accumulation resulted in reduced carotenoid content, demonstrating a trade-off between these pigment groups.

Fig. 7.

A-C Carotenoids to chlorophylls ratio in Dunaliella strains B8, G23, and I44 under various environmental conditions, highlighting the effects of light intensity, salinity, nitrate, copper, and harvesting time on biomass production. Treatments are abbreviated HL  High Light (1200 µmol m⁻² s⁻¹), LL  Low Light (200 µmol m⁻² s⁻¹), HS  High Salinity (3 M), LS  Low Salinity (1 M), HN  High Nitrate (0.5 M), LN  Low Nitrate (0 M), HC  High Copper (0.05 mM), LC  Low Copper (0 mM). Panels correspond to strains as follows: A, B8; B, G23; C, I44

The light regime had a major impact on chlorophyll content. Maximum chlorophyll accumulation was observed under low-light, low salinity, high nitrate, and no copper supplementation at extended cultivation for 14 days (Fig. 5A). Under these conditions, strain G23 achieved a chlorophyll concentration of 31.47 ± 0.6 mg L⁻¹, followed by strain B8 (30.10 ± 0.7 mg L⁻¹) and strain I44 (20.51 ± 0.5 mg L⁻¹).

High light and nutrient-enriched conditions resulted in lower chlorophyll levels and increased carotenoid accumulation, indicating a shift in pigment composition. Under high light, low salinity, high nitrate, and high copper supplementation, chlorophyll levels remained consistently lower than in the corresponding control conditions. For example, in strain B8 exposed to a high light, low salinity, high nitrate, and high copper, chlorophyll content rose from 2.27 mg L⁻¹ on day 0 to 9.96 mg L⁻¹ on day 14, whereas the control reached 29.06 mg L⁻¹. Similar patterns were observed in strain G23 (8.11 mg L⁻¹ vs. 30.07 mg L⁻¹) and strain I44 (4.12 mg L⁻¹ vs. 20.51 mg L⁻¹).

Salinity had a significant effect on chlorophyll accumulation. High salinity conditions strongly reduced chlorophyll content in all strains, whereas low-salinity conditions supported higher chlorophyll levels when combined with optimal light and sufficient nitrogen availability. Nitrogen availability was also critical, as treatments without nitrate supplementation showed much lower chlorophyll levels.

Copper supplementation (0.05 mM CuSO₄·7 H₂O) was associated with a slight reduction in chlorophyll content alongside enhanced carotenoid accumulation in all strains. The trade-off between chlorophyll and carotenoid production was most evident at earlier cultivation (day 7), whereas extended cultivation to day 14 resulted in partial chlorophyll recovery accompanied by reduced carotenoid levels. A summary of the treatment conditions associated with maximum biomass and accumulation of lutein, β-carotene, and chlorophyll is provided in Table 5.

Table 5.

Culture conditions associated with maximum biomass, lutein, β-carotene, and chlorophyll accumulation within the tested experimental ranges

strain	Response	Light	NaCl	Nitrate	Copper	Harvest time		Maximum value
B8	Biomass	High(1200)	1 M	0.5 M	0.05 mM		14	1.58 g L⁻¹
G23	Biomass	High(1200)	1 M	0.5 M	0.05 mM		14	1.51 g L⁻¹
I44	Biomass	High(1200)	1 M	0.5 M	0.05 mM		14	1.46 g L⁻¹
B8	Lutein	High(1200)	1 M	0.5 M	0.05 mM		7	11.5 mg g⁻¹ DW
G23	Lutein	High(1200)	1 M	0.5 M	0.05 mM		7	7.3 mg g⁻¹ DW
I44	Lutein	High(1200)	1 M	0.5 M	0.05 mM		7	6.5 mg g⁻¹ DW
B8	β-Carotene	High(1200)	1 M	0.5 M	0.05 mM		7	63.5 mg g⁻¹ DW
G23	β-Carotene	High(1200)	1 M	0.5 M	0.05 mM		7	41.5 mg g⁻¹ DW
I44	β-Carotene	High(1200)	1 M	0.5 M	0.05 mM		7	51.6 mg g⁻¹ DW
B8	Chlorophyll	Low(200)	1 M	0.5 M	0 mM		14	30.10 mg L⁻¹
G23	Chlorophyll	Low(200)	1 M	0.5 M	0 mM		14	31.47 mg L⁻¹
I44	Chlorophyll	Low(200)	1 M	0.5 M	0 mM		14	20.51 mg L⁻¹

Light is given as photon flux density (µmol photons m⁻² s⁻¹); NaCl and nitrate concentrations are expressed as molarity (NaCl, KNO₃); copper as CuSO₄·7 H₂O. Biomass values are given as g L⁻¹, lutein and β-carotene as mg g⁻¹ DW, and chlorophyll as mg L⁻¹

Discussion

The findings indicate that indigenous Dunaliella strains are capable of sustaining both growth and pigment production under elevated light intensity, reflecting their inherent stress acclimation capacity as reported in previous studies [24]. In particular, strains B8, G23, and I44 exhibited enhanced biomass accumulation alongside increased pigment levels under high light, suggesting a coordinated metabolic adjustment to high light exposure [24]. Although significant genotype effects on biomass were detected, the relatively small magnitude of inter-strain variation implies that environmental drivers, particularly light conditions, played a more dominant role in growth regulation than genetic background, consistent with reports highlighting the primary influence of culture conditions such as light and salinity on Dunaliella performance [47].

High-light regime exposure likely triggered metabolic adjustments associated with increased carotenoid biosynthesis, particularly lutein and β-carotene, potentially via enhanced activity of the plastidial MEP pathway, as suggested by recent studies on isoprenoid and carotenoid metabolism in photosynthetic organisms [27, 48, 49]. However, in the present study, ROS levels and MEP pathway activity were not directly measured, and our interpretation of these mechanisms is therefore inferred from earlier physiological and molecular analyses in Dunaliella and related microalgae, which have shown ROS-induced upregulation of carotenogenic pathways under excess irradiance or oxidative stress [7, 24, 49]. The accumulation of these pigments is generally interpreted as a photoprotective response to elevated reactive oxygen species (ROS) generated under high irradiance, rather than as evidence of intrinsic high light resistance [7, 49, 50].

Salinity exerted a significant influence on both growth and pigment accumulation. Low-salinity conditions consistently supported higher biomass and pigment content compared to high-salinity conditions. These results align with previous studies showing that Dunaliella and other microalgae perform optimally at moderate salinity, whereas extreme salinity imposes osmotic and ionic stress that constrains cellular water balance, photosystem stability, and overall photosynthetic efficiency, ultimately limiting growth and pigment biosynthesis [35, 51–54]. The observed reduction in chlorophyll content under combined high light and high-salinity conditions indicates a stress-induced shift in pigment composition, with carotenoid accumulation accompanying the decline in chlorophyll. Our results revealed an inverse relationship between chlorophyll and carotenoid content, consistent with a stress-induced pigment shift reported in Dunaliella strains under high light and salinity [35, 51, 52] .This finding underscores the photoprotective role of carotenoids as chlorophyll declines. The shift likely reflects a photoacclimatory adjustment, whereby carotenoids partially compensate for chlorophyll loss under elevated osmotic and irradiance stress. Such a pattern has also been observed in other Dunaliella strains where high salinity and irradiance promote carotenoid enrichment at the expense of chlorophyll [35, 51, 52].

Nitrogen availability was a key factor coordinating both biomass production and carotenoid accumulation in our Dunaliella isolates. Severe nitrogen limitation classically reduces protein and chlorophyll synthesis, suppresses photosynthetic activity, and creates an imbalance between carbon fixation and nitrogen assimilation, redirecting carbon toward secondary carotenoid biosynthesis via ROS-mediated signaling [7, 55–57]. In contrast, nitrate-replete conditions maintained photosynthetic capacity, allowing moderate stressors such as high light and salinity to stimulate carotenoid biosynthesis without substantial growth inhibition [54, 58, 59]. This highlights a productivity-oriented metabolic regime in which sufficient nitrogen supports balanced carbon partitioning between biomass formation and secondary metabolism, resulting in higher volumetric carotenoid yield compared with nitrogen-limited cultures [58–60]. Our findings suggest that moderate stress under adequate nutrient availability can maximize carotenoid productivity, providing practical insights for Dunaliella cultivation strategies [54, 59, 61].

Copper supplementation affected pigment accumulation, with low copper supplementation enhancing both biomass and carotenoid production, especially in strain B8, while control cultures without added copper showed lower pigment levels. Copper functions as a cofactor in several redox-related enzymes and has been reported to modulate microalgal pigment and carotenoid biosynthesis in a dose-dependent manner, whereby trace to low micromolar levels can stimulate photosynthesis and pigment accumulation, whereas higher concentrations become inhibitory to growth and photosynthetic efficiency [26, 62, 63]. In line with these observations, our data indicate that low copper supplementation acted as a mild stress that supported pigment accumulation without markedly suppressing biomass, and we therefore refrain from extrapolating any general “higher copper inhibitory” effects beyond the tested range [26, 62].

The lutein (up to 11.5 mg g⁻¹ DW) and β-carotene (up to 63.5 mg g⁻¹ DW) contents achieved in this study are within the upper ranges reported for well-characterized.

Dunaliella strains under stress conditions, confirming the strong carotenogenic potential of these indigenous isolates. When expressed on a volumetric basis, the maximum β-carotene concentrations (up to ~ 40 mg L⁻¹ in B8) and lutein concentrations (up to ~ 7 mg L⁻¹ in B8) are comparable to values reported for productive D. salina cultures operated in outdoor ponds or two-stage photobioreactor systems designed for β-carotene enrichment [11, 17, 57, 64–66].

Under moderate stress conditions, including high light and low salinity, sufficient nitrate availability maintains photosynthetic efficiency and carbon fixation, enabling partial diversion of carbon flux toward secondary carotenoid biosynthesis without substantially inhibiting biomass accumulation [58, 59, 67]. Reactive oxygen species (ROS) act as signaling molecules promoting carotenoid biosynthesis under moderate stress [58, 67]. From a bioprocess perspective, the divergence between conditions that maximize biomass and those that enhance carotenoid content represents a fundamental biomass–carotenoid trade-off [12, 54, 59]. Our results show that short-term exposure to high light, low salinity, high nitrate, and low copper supplementation (day 7) is optimal for carotenoid enrichment, whereas prolonged cultivation (day 14) under the same conditions favors biomass accumulation but leads to pigment dilution and partial degradation, mirroring the two-phase cultivation concept widely applied in Dunaliella β-carotene production [51, 57, 66, 68]. This implies that economically viable Dunaliella-based production schemes will likely require either two-stage cultivation (a growth phase followed by a stress-induction phase) or carefully optimized one-stage regimes that balance growth and stress to achieve an acceptable compromise between biomass yield and carotenoid productivity [12, 31, 69, 70].

These results indicate that precise modulation of light regime, salinity, nitrate availability, and copper supplementation can facilitate the co-production of biomass and high-value carotenoids in indigenous Dunaliella strains, offering valuable insights for sustainable microalgal production strategies and positioning these isolates within the broader spectrum of Dunaliella-based processes reported in recent literature [36, 68, 69, 71].

Conclusions

This study demonstrates that indigenous Dunaliella isolates, particularly B8, possess strong stress-induced carotenogenic potential, achieving up to 11.5 mg g⁻¹ DW lutein and 63.5 mg g⁻¹ DW β-carotene with a maximum biomass of 1.58 g L⁻¹. Clear strain-specific responses and a biomass–carotenoid trade-off were observed. It was shown that the short-term high light exposure under low salinity with adequate nitrate and copper maximized carotenoid accumulation, whereas prolonged cultivation primarily promoted biomass. These findings highlight the importance of precisely modulated environmental and nutrient conditions for sustainable carotenoid production. Future studies should focus on scaling these optimized regimes to pilot- and industrial-scale photobioreactors, evaluating one- versus two-stage cultivation strategies, and integrating metabolomic and transcriptomic analyses to clarify regulatory mechanisms. Testing these strains under region-specific outdoor conditions will be critical for translating laboratory findings into commercially viable microalgal production systems.

Authors’ contributions

**AB and AH** conceived and designed the study. **MM** performed the experiments and data analysis. **MM** prepared the graphs and wrote the manuscript. **BP** contributed to manuscript writing. The author(s) read and approved the final manuscript.

Funding

The financial support of the University of Tabriz and the Agricultural Biotechnology Research Institutes of Iran is gratefully acknowledged.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All experimental studies on plants were conducted in compliance with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

Not applicable.