Comprehensive Growth Analysis to Detect Optimal Time Point for Carotenoid Production: A Comparative Study in Spirulina subsalsa and Chlorella pyrenoidosa

Abstract

This study investigated the growth dynamics of Spirulina subsalsa and Chlorella pyrenoidosa under standardized phototrophic cultivation conditions to determine the optimal harvest time for maximum carotenoid yield. Both species were maintained at temperature (28℃-35℃), relative humidity (52%-55%), light intensity (2.5-3.5 klx) and a photoperiod of (10:14 h) light/dark cycle. Growth dynamics were assessed by monitoring cell density, biomass productivity, protein content, chlorophyll and carotenoid content over a defined cultivation period. S. subsalsa showed rapid growth, reaching peak cell density with in a shorter duration of 12 days, compared to C. pyrenoidosa which showed longer duration of 27 days. The biomass productivity was higher (186.36 ± 0.029 mg/L/day) in Spirulina compared to Chlorella (12.98 ± 0.546 mg/L/day). S. subsalsa showed higher protein content on day 6 (33.634 ± 0.97 mg/g fresh weight (FW)), higher chlorophyll on day 8 (1.25 ± 0.034 mg/g FW) and higher carotenoid on day 12 (0.379 ± 0.003 mg/g FW) compared to C. pyrenoidosa with maximum protein (3.374 ± 0.039 mg/g FW), maximum chlorophyll (0.252 ± 0.002 mg/g FW) and maximum carotenoid (0.054 ± 0.001 mg/g FW) on day 27. The protein, chlorophyll, and carotenoid levels showed decrease in both the algae at the end of the experiment. Harvesting S.subsalsa on day 12 and C. pyrenoidosa on day 27 will result in increased carotenoid yield and enhance the efficiency of carotenoid extraction. The study provides a base for sustainable large-scale cultivation of two microalgal species with targeted harvesting of natural carotenoids to meet industrial demand.

Keywords:
microalgae; Spirulina subsalsa; Chlorella pyrenoidosa; standardized microalgal cultivation; comparative microalgal study; microalgal growth analysis; time-dependent metabolite synthesis; carotenoid production peak; optimal harvest time.

HIGHLIGHTS

Maximum carotenoids are observed on day 12 for Spirulina subsalsa

Maximum carotenoids are observed on day 27 for Chlorella pyrenoidosa

Spirulina shows faster growth than Chlorella with slow and sustained growth

Total protein, chlorophyll and carotenoids are higher in Spirulina than Chlorella

INTRODUCTION

Microalgae are rich in nutrients such as proteins, enzymes, fatty acids, minerals, vitamins, and pigments like carotenoids. Among these metabolites, carotenoids have selectively proved to be beneficial for human health, particularly in preventing chronic diseases [¹]. Microalgae have been shown to produce a variety of carotenoids, including xanthophylls and carotenes, as a part of their normal growth metabolism [²]. Carotenoids like β-carotene, zeaxanthin, and lutein show strong antioxidant properties against reactive oxygen species (ROS), by protecting cells from oxidative damage [³-⁵]. Both raw extracts and purified β-carotene obtained from microalgae are observed to show immunomodulation, hepatoprotective activity, anti-inflammatory properties, anti-obesity and anti-cancer effects [⁶-¹⁰]. Xanthophylls like lutein and zeaxanthin play a role as macular pigments and accumulate in the cornea to protect the retinal membrane from the damage caused by blue light supporting visual clarity. Lutein pigment is also found to help with health conditions like Alzheimer’s disease, diabetic retinopathy, retinal nerve diseases, cataracts and age-related macular degeneration [⁶,¹¹,¹²]. Both chlorophyll and carotenoids are in high commercial demand. Chlorophyll plays its role as a natural colorants in food and pharmaceuticals. Other health promoting properties of chlorophylls include prevention of diabetes, viral and chronic inflammatory diseases, cardiovascular diseases, metabolic syndrome, tumors, nerve damage and ophthalmic diseases [¹³-¹⁵].

The global market value of carotenoids in 2024 was approximately $2.1 billion USD as per the BCC Research reports and is expected to increase to $2.9 billion USD by 2029 at a compound annual growth rate (CAGR) of 6.7% [¹⁶]. Though, there are large applications of carotenoids, the carotenoid market is largely dominated by chemically synthesized carotenoids contributing 80-90% where as 10-20% of carotenoids are derived from natural resources [¹⁷,¹⁸]. The rising awareness of consumers regarding food safety and their preference for plant derived bioactive compounds are increasing the demand for natural carotenoids [¹⁷].

Microalgae possess several advantages for carotenoid production such as lower nutrient requirements, shorter growth cycles and higher photosynthetic efficiency when compared to terrestrial plants [¹⁹]. Additionally, the synthesis of carotenoids (carotenogenesis), is stimulated by the presence of reactive oxygen species (ROS), produced under various stress conditions such as exposure to high light intensity, salinity, or high temperatures [⁶].

Though, microalgae shows huge biodiversity, only a few species have been investigated extensively for biotechnological applications [²⁰-²²]. Among these, the most studied are the species of Arthrospira (Spirulina) platensis and Chlorella vulgaris. Several studies have reported the growth performance of different species of Spirulina and Chlorella under varying environmental conditions [²³,²⁴] It is necessary to identify and explore underutilized microalgal species such as S. subsalsa and C. pyrenoidosa to discover and optimize alternative sources for commercially valuable carotenoids and to detect novel bioactive compounds.

S. subsalsa is a cyanobacteria belonging to the order Oscillatoriales, which are found to be spiral, or coiled filamentous in morphology [²⁵]. Some studies have investigated its ability to produce different bioactive metabolites and its role as a bioremediation agent [²⁶,²⁷]. Previous studies showed that high light intensity increased the production of antioxidant compounds like carotenoids and polyphenols in S. subsalsa [²⁰]. A study also reported that, S. subsalsa attained optimal growth at 35℃ with increased production of phycocyanin under green light [²⁸]. C. pyrenoidosa is a single-celled, spherically shaped, fresh water growing green algae [²⁹]. Previous findings acknowledge C. pyrenoidosa as a third-generation bioenergy source due to its remarkable photosynthetic efficiency [³⁰]. C. pyrenoidosa is widely used for its various health benefits such as anti-inflammatory, antioxidant, anti-bacterial, anti-tumor and immune boosting properties due to its rich nutrient profile [³⁰,³¹]. The rich carotenoid content of C. pyrenoidosa was found to play a major role in its antitumour activity [³²].

Growth medium composition plays an important role on microalgal biomass production and accumulation of bioactive metabolites as variations based on nutrient supply significantly affects the carotenoid and chlorophyll pigment production. An increase in nitrate concentration in the growth medium was reported to boost β-carotene production in S. platensis, when grown under specific environmental conditions [³³]. There are no similar studies on S. subsalsa. Research on optimization of lutein production in C. pyrenoidosa included improved lutein biosynthesis using sodium bicarbonate (100-150 mM) for its cultivation as well as exposing the algae to a condition of nutrient starvation [³⁴,³⁵]. Extraction methods have been further developed to optimize yields from microalgal biomass. These include non-mechanical methods like thermal, chemical and enzymatic methods and mechanical methods such as pressurized systems, electric fields, microwave, ultrasonication and supercritical extraction [³⁶]. Lutein and chlorophyll recovery was optimized using pulsed electric field (PEF)-assisted extraction [³⁷]. However, large-scale, cost-effective extraction remains a challenge. The extraction of Spirulina and Chlorella biomass requires individual optimization due to its diverse growth characteristics. Before proceeding with analysis of carotenoid profiles of these algae, one would need to develop an efficient carotenoid extraction procedure. Identifying the optimal time point of carotenoid production during the growth cycle can boost carotenoid extraction and hence, is a primary step before starting the extraction process.

While there are studies on different carotenoids present in various microalgae, there is no comprehensive data on the full spectrum of carotenoids or their isomers in S. subsalsa and C. pyrenoidosa. Most existing research is either on single species of algae or different algal species belonging to same group, lacking comparative studies across different algal groups. The present study provides a comparative growth analysis of S. subsalsa (blue-green algae) and C. pyrenoidosa (green algae) to determine the optimal time of carotenoid accumulation. Both algae were cultivated under controlled conditions of temperature (28℃-35℃), relative humidity (52%-55%), and light intensity (2.5-3.5 klx), using different optimal growth medium for each alga. Zarrouk’s culture medium was used for S. subsalsa and modified Bold’s Basal Medium (BBM) for C. pyrenoidosa. Growth and biomass productivity of both the algae were assessed at various time points. Important biochemical parameters, including protein, chlorophyll, and carotenoid content, were also measured at specific intervals. The growth period promoting maximum carotenogenesis in both the algae was identified by quantifying the carotenoids produced during specific growth intervals using spectrophotometric assays.

MATERIAL AND METHODS

Media composition

The strain NCIM-5143 of S.subsalsa and NCIM-2738 of C. pyrenoidosa were obtained from the culture collection center of National Chemical Laboratory (NCL), Pune, India. Both the cultures were cultivated phototrophically. S. subsalsa was cultivated in Zarrouk’s culture medium with following composition (g/L): NaHCO₃-16.8 g, NaNO₃-2.5 g, K₂HPO₄-0.5 g, K₂SO₄-1.0 g, NaCl-1.0 g, CaCl₂.2H₂O-0.04 g, MgSO₄.7H₂O-0.2 g, FeSO₄7H₂O-0.01 g and 1.0 mL of A5 solution which was composed of H₃BO₃-2.86 g, MnCl₂4H₂O-1.81 g, ZnSO₄4H₂O-0.222 g, Na₂MoO₄-0.018 g, CuSO₄5H₂O-0.079 g [³⁸,³⁹]. C. pyrenoidosa was cultivated in modified Bold’s Basal Medium (BBM) with following composition (g/L): KNO₃-0.375 g, K₂HPO₄-0.08 g, MgSO₄.7H₂O-0.1 g, CaCl₂.2H₂O-0.0375 g, NaCl-0.015 g, FeSO₄-0.005 g, Na₂EDTA-0.0625 g [⁴⁰].

Preparation of stock cultures of algal samples

Agar slopes of S. subsalsa NCIM-5143 and C. pyrenoidosa NCIM-2738 were obtained from National Chemical Laboratory (NCL) culture collection center. A loopful of Spirulina and Chlorella culture samples were transferred into 20 mL of fresh Zarrouk’s culture medium and modified Bold’s Basal Medium (BBM) respectively, in Erlenmeyer flasks (100 mL) each under aseptic conditions. The culture inoculums were scaled up every two weeks. The final culture volumes for S. subsalsa and C. pyrenoidosa were raised to 2 liters and 4 liters, respectively, using larger 3-liter and 5-liter glass bottles. Final volume of stock cultures were decided considering the maintenance and growth rates of both the algal samples. For the maintenance of stock cultures, a photobioreactor (dimensions = 33 cm width x 47 cm height x 43 cm breadth) was constructed to provide optimal growth conditions and a simple cost-effective agitator system (speed = 60 rpm) was developed using silicon bottle brush for efficient mixing of the culture medium. The main body of the photobioreactor was constructed using acrylic sheet (thickness = 3 mm) and the basal part consisted of aluminum sheet. Illumination was provided using natural white light LED strips. Four LED strips (5-meter long) of natural white light were used to surround the photobioreactor, with each strip interconnected in series. The LED strips were equally spaced to support uniform lighting (Figure 1). The growth conditions were maintained in the following range., temperature (28℃-35℃), relative humidity (52%-55%), and light intensity (2.5-3.5 klx) throughout the experimental period [²⁴,⁴¹-⁴⁴]. The cultures were maintained under a photoperiod of 10/14 hours (light/dark cycle) against the conventional 12/12 hour light/dark cycle, due to the institutes operating hours. Temperature, humidity, and light intensity were checked two times (morning and evening) daily during the experiment using thermometer, humidity meter, and lux meter (Lutron LX-101A), respectively.

Figure 1
Lab-scale photobioreactor setup for the phototrophic cultivation of S. subsalsa and C. pyrenoidosa under controlled light and temperature conditions.

Preparation of experimental inoculum

The culture inoculum of S. subsalsa and C. pyrenoidosa were prepared and maintained in the photobioreactor to acclimate the cultures for growth curve experiment. The culture medium for both the algae was prepared by dissolving each chemical separately and then mixing them together to make a final volume of 1000 mL. This step was followed by sterilization of the culture medium using nylon membrane filters (size-0.2 μm, 47 mm). For the preparation of culture inoculum, 100 mL of the culture samples from Spirulina and Chlorella stock solutions were transferred into 1000 mL of fresh sterile Zarrouk’s medium and modified BBM, respectively. The inoculum cultures were cultivated for about two weeks. For further growth curve experiments, these prepared live stocks were used as the inoculum. The experimental inoculums were prepared using multiple Erlenmeyer flasks (500 mL) each, by transferring 20 mL of the inoculum in to 200 mL of freshly prepared sterilized media. From these multiple flasks, a single flask was used to measure all the selected growth parameters per day to reduce the risk of sample contamination. The flasks were checked for similarity in size to reduce variability in growth outcomes. The consistency of the inoculum samples in multiple flasks were confirmed by measuring optical density (OD) at 680 nm. Manual agitation of the inoculum cultures were performed twice (morning and evening) everyday.

Growth analysis of S. subsalsa and C. pyrenoidosa

Important growth parameters like cell density, biomass productivity, total protein and pigment content (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid) were assessed on specific days. Measurements were taken on days (2, 4, 6, 8, 10, 12, and 14) for S. subsalsa and days (5, 10, 15, 20, 23, 25, 27, and 31) for C. pyrenoidosa. The days for analysis were selected based on the preliminary growth curve experiment results which were performed before standardizing the final protocols.

Determination of microalgal growth

Measurement of S. subsalsa and C. pyrenoidosa growth was performed by recording absorbance at 680 nm with the help of a UV/Vis spectrophotometer (Equiptronics EQ 822A) [⁴⁵,⁴⁶]. The cell concentration was determined for C. pyrenoidosa by cell count method using the Neubauer hemocytometer [⁴⁷,⁴⁸]. As S. subsalsa appears filamentous, traditional methods of cell count were not applicable. To address this, the trichome density of S. subsalsa for every experimental day was assessed by visually observing under a light microscope. Spirulina sample volume (10 μL) used to examine trichome density per experimental day was kept constant. The sample was well mixed prior to the analysis to avoid clumping and ensure uniformity. The images of Spirulina sample observed under the light microscope (40x magnification) were captured to document the trichome density. The dry weight (initial and final) of both the algae was measured to determine the biomass productivity at the end of the growth curve experiment (14^th day - Spirulina and 31^st day - Chlorella) [⁴⁹,⁵⁰]. Biomass productivity was calculated by using values for initial dry weight and final dry weight. Initial dry weight was determined on the day of experimental inoculation (day 0) by filtering 20 mL of inoculum samples (S. subsalsa and C. pyrenoidosa) through pre-weighed Whatman No. 3 filter paper followed by its rinsing with distilled water. Further process involved oven-drying (60°C) of the wet biomass until a constant weight was achieved. After desiccation of the dry biomass on pre-weighed filter paper, the dry weight of the samples were measured. This process was repeated at the end of growth experiment to measure the final dry weight of algal samples. The volumetric biomass productivity (P_Biomass) of algae was calculated [⁵¹].

Protein extraction and quantification

Total protein content was determined using the Lowry assay [⁵²]. Bovine serum albumin (BSA) at a concentration range of 0.0625 to 2 mg/mL was used to prepare a calibration curve. The protein concentrations of S. subsalsa and C. pyrenoidosa for each experimental day were determined by plotting graphs of (absorbance against protein concentration) and the values were expressed as mg/g fresh weight (FW). For protein analysis, 0.1 g of the fresh Spirulina biomass was used. For protein analysis in Chlorella, initial tests with 0.1 g of fresh biomass showed absorbance values against the readable range. As a result, 0.5 g of fresh Chlorella biomass was used for the final experimental analysis to ensure accurate results. The algal samples were crushed in 3 mL of 0.1N NaOH using mortar and pestle. Subsequent mixture was centrifuged for 30 min at 2000 rpm and the supernatant was collected. After transferring 1 mL of the supernatant solution in to a 20 mL test tube, 5 mL of alkaline copper solution was added to it. The resulting solutions were left for incubation for 10 minutes at room temperature. This step was followed by addition of 0.5 mL Folin-Ciocalteu reagent (1:1 diluted). After incubating the mixture for 30 minutes, the values for absorbance were measured at 750 nm using a UV/Vis spectrophotometer (Equiptronics EQ 822A).

Pigment extraction and quantification

Arnon’s method was used to quantify pigments from S. subsalsa and C. pyrenoidosa for each experimental day and the values were expressed as mg/g fresh weight (FW) [⁵³]. The amount of fresh biomass used for pigment analysis for both the algae was consistent with the amount used for protein analysis. 0.1 g of Spirulina and 0.5 g of Chlorella fresh biomass was crushed in 95% ethanol using mortar and pestle and an ice bath. The resulting mixture was centrifuged at room temperature (27°C-30°C) for 30 minutes at 2000 rpm. The collected supernatant was checked for absorbance at the following wavelengths (480 nm, 510 nm, 645 nm, and 663 nm) using a UV/Vis spectrophotometer (Equiptronics EQ 822A). Pigment content was calculated using the following formula: Chlorophyll a (Chl_a) mg/g FW= 12.7 x A₆₆₃ - 2.69 x A₆₄₅ x volume of sample (mL) / 1000 x weight of tissue (g); Chlorophyll b ( Chl_b) mg/g FW= 22.9 x A₆₄₅ - 4.68 x A₆₆₃ x volume of sample (mL)/ 1000 x weight of tissue (g); Total Chlorophyll (Chl_total) mg/g FW= 20.21 x A₆₄₅ + 8.02 x A₆₆₃ x volume of sample (mL) / 1000 x weight of tissue (g); Total Carotenoids (Car_total) mg/g FW = 7.6 x A₄₈₀ - 1.49 x A₅₁₀ x volume of sample (mL)/ 1000 x weight of tissue (g).

Statistical analysis

Data were analysed statistically using Microsoft Excel for standard deviation (SD) and standard error (SE) calculations. Python (SciPy library) was used for statistical tests. Experiments were carried out in triplicates (n=3). The results were reported as mean ± SE at 95% confidence. Differences between time points were analyzed using one-way analysis of variance (ANOVA), while pairwise comparisons were assessed using an independent t-test, with statistical significance set at p < 0.05.

RESULTS

Comparison of growth patterns of S. subsalsa and C. pyrenoidosa using turbidity and cell count measurements

Comparative growth analysis of S. subsalsa and C. pyrenoidosa was studied. Figure 2 presents the comparative growth analysis of S. subsalsa and C. pyrenoidosa against time, using turbidity by noting optical density (OD) measurements at 680 nm. Growth of S. subsalsa was also checked by observing trichome density on every experimental day. The images were captured after every visual inspection to document the trichome density of S. subsalsa for each experimental day as shown in Figure 3. Results of both methods concluded that S. subsalsa shows rapid growth within 14 days. The highest growth in S. subsalsa was observed on day 12 with a maximum OD of 2 ± 0.025. A lag phase was observed in S. subsalsa during days 0 to 2 followed by an exponential phase during days 4 to 10. During days 12 to 14, growth was found to slow down with relatively stable OD, indicating stationary phase.In comparison, C. pyrenoidosa showed slower but sustained growth up to 31 days. The highest growth in C. pyrenoidosa was observed on day 31 with a maximum OD of 0.995 ± 0.017. C. pyrenoidosa growth as analyzed for every experimental day using turbidity showed a stable increase in cell density from days 5 to 31. C. pyrenoidosa growth was also analyzed for every experimental day using cell count measurements. However, the cell count results for C. pyrenoidosa was maximum (6.25 x 10⁶ ± 14.974 cells/mL) on day 27 followed by a decline on day 31 as seen in Table 1 and Figure 4. The difference in turbidity and cell count data of C. pyrenoidosa indicates interference of other factors like cell aggregation, release of extracellular substances, and cell debris to overall turbidity increase detected at the final day i.e. day 31 of Chlorella growth experiment. As a result, based on the cell count data, it can be concluded that C. pyrenoidosa strain NCIM 2738 shows a decline in its growth on day 31 of the growth experiments. Comparing the experimental results obtained for both the algae, it can be concluded that S. subsalsa shows rapid growth compared to C. pyrenoidosa.

Table 1
Growth analysis of C. pyrenoidosa: Cell count v/s Time.

Time (Day)	Cell Count (x 106 cells/mL)
0 5 10 15 20 23 25 27 31	0.683 ± 0.038 2.405 ± 0.047 3.913 ± 0.034 4.368 ± 0.044 4.515 ± 0.047 5.048 ± 0.044 5.750 ± 0.055 6.250 ± 0.037 4.335 ± 0.038

Figure 2
Comparative growth analysis of S. subsalsa and C. pyrenoidosa: Optical Density v/s Time. The data are the mean values of 3 replicates ± SE at 95% confidence. Statistical significance was determined using an independent t-test. A single asterisk (*) indicates a statistically significant difference (p < 0.05) between species.

Figure 3
Growth analysis of S. subsalsa using images capturing trichome density under a light microscope (40x magnification) on different days (A, B, C, D, E, F and G corresponding to day 2, 4, 6, 8, 10, 12 and 14 respectively). These images represent multiple observations.

Figure 4
Growth analysis of C. pyrenoidosa: Cell count v/s Time. The data are the mean values of 3 replicates ± SE at 95% confidence. Statistical analysis was performed using One-Way ANOVA. Three asterisks (***) indicate a highly significant difference (p < 0.001) between time points.

Comparison of biomass productivity (mg/L/day) in S. subsalsa and C. pyrenoidosa.

The growth of S. subsalsa and C. pyrenoidosa were also assessed by estimating the biomass productivity during the cultivation period as shown in Table 2. The biomass productivity was found to be higher in S. subsalsa (186.36 ± 0.029 mg/L/day) throughout the 14-day cultivation period compared to C. pyrenoidosa (12.98 ± 0.546 mg/L/day) throughout the 31-day cultivation day.

Table 2
Comparative Analysis of Biomass Productivity (mg/L/day) in S. subsalsa and C. pyrenoidosa.

Algal species	Biomass productivity (mg/L/day)
S. subsalsa	186.360 ± 0.029
C. pyrenoidosa	12.980 ± 0.546

Comparison of protein (mg/g FW), chlorophyll (mg/g FW), and carotenoid (mg/g FW) contents in S. subsalsa and C. pyrenoidosa.

The total protein, total chlorophyll and total carotenoid contents were found to be considerably higher in S. subsalsa than C. pyrenoidosa during the experimental period as seen in Table 3. In S. subsalsa, the total protein content was maximum (33.634 ± 0.97 mg/g FW) on day 6 as shown in Figure 5, the total chlorophyll was maximum (1.25 ± 0.034 mg/g FW) on day 8 and the total carotenoid was maximum (0.379 ± 0.003 mg/g FW) on day 12 as given in Figure 6. In comparison, C. pyrenoidosa showed maximum amount of total protein (3.374 ± 0.039 mg/g FW) as seen in Figure 5. Maximum total chlorophyll (0.252 ± 0.002 mg/g FW) and total carotenoid (0.054 ± 0.001 mg/g FW) were also found to be present on day 27 in C. pyrenoidosa as seen in Figure 7. Towards the completion of growth cycle, the protein, chlorophyll and carotenoid contents were found to decline in both the algal species studied. Comparative analysis of changes in pigment contents over time in S. subsalsa and C. pyrenoidosa is shown in Figure 8.

Table 3
Comparison of Protein (mg/g FW), Chlorophyll (mg/g FW), and Carotenoid (mg/g FW) contents in S. subsalsa and C. pyrenoidosa.

Algal species		Time (Day)	Protein (mg/g FW)	Chl a (mg/g FW)	Chl b (mg/g FW)	Total Chl (mg/g FW)	Total Car (mg/g FW)
S. subsalsa		2 4 6 8 10 12 14	9.192 ± 0.568 15.237 ± 0.784 33.634 ± 0.970 26.749 ± 1.131 22.685 ± 0.710 14.061 ± 0.248 9.827 ± 0.316	0.151 ± 0.006 0.222 ± 0.006 0.521 ± 0.009 0.746 ± 0.013 0.704 ± 0.036 0.489 ± 0.001 0.270 ± 0.005	0.190 ± 0.002 0.266 ± 0.014 0.396 ± 0.020 0.504 ± 0.040 0.428 ± 0.049 0.286 ± 0.011 0.254 ± 0.009	0.342 ± 0.008 0.487 ± 0.020 0.917 ± 0.011 1.250 ± 0.034 1.132 ± 0.014 0.775 ± 0.011 0.524 ± 0.014	0.065 ± 0.014 0.178 ± 0.003 0.204 ± 0.003 0.265 ± 0.005 0.320 ± 0.004 0.379 ± 0.003 0.180 ± 0.002
C. pyrenoidosa		5 10 15 20 23 25 27 31	1.174 ± 0.092 1.464 ± 0.024 1.866 ± 0.075 2.109 ± 0.075 2.636 ± 0.076 3.247 ± 0.016 3.374 ± 0.039 1.720 ± 0.069	0.007 ± 0.000 0.013 ± 0.001 0.029 ± 0.001 0.046 ± 0.001 0.078 ± 0.001 0.134 ± 0.001 0.124 ± 0.001 0.032 ± 0.001	0.009 ± 0.002 0.012 ± 0.001 0.022 ± 0.001 0.044 ± 0.001 0.075 ± 0.001 0.112 ± 0.006 0.128 ± 0.001 0.032 ± 0.002	0.015 ± 0.002 0.025 ± 0.002 0.050 ± 0.001 0.090 ± 0.000 0.153 ± 0.002 0.245 ± 0.005 0.252 ± 0.002 0.064 ± 0.001	0.005 ± 0.000 0.010 ± 0.000 0.015 ± 0.000 0.030 ± 0.000 0.046 ± 0.000 0.053 ± 0.002 0.054 ± 0.001 0.026 ± 0.000

Figure 5
Comparative protein content (mg/g FW) in S. subsalsa and C. pyrenoidosa. The data are the mean values of 3 replicates ± SE at 95% confidence. Statistical significance was determined using an independent t-test. Three asterisks (***) indicate a highly significant difference (p < 0.001) between species.

Figure 6
Changes in the following pigment contents (mg/g FW): Chlorophyll a, Chlorophyll b, Total Chlorophyll and Total carotenoid in S. subsalsa. The data are the mean values of 3 replicates ± SE at 95% confidence.

Figure 7
Changes in the following pigment contents (mg/g FW): Chlorophyll a, Chlorophyll b, Total Chlorophyll and Total carotenoid in C. pyrenoidosa. The data are the mean values of 3 replicates ± SE at 95% confidence.

Figure 8
Comparative pigment contents (mg/g FW): Total Chlorophyll and Total carotenoid in S. subsalsa and C. pyrenoidosa. The data are the mean values of 3 replicates ± SE at 95% confidence. Statistical significance was determined using an independent t-test. Three asterisks (***) indicate a highly significant difference (p < 0.001) between species.

DISCUSSION

In this study, we observed distinct growth patterns and biochemical compositions in S. subsalsa NCIM 5143 and C. pyrenoidosa NCIM 2738. The turbidity of S. subsalsa showed a rapid increase, peaking on day 12, indicating a faster growth rate compared to C. pyrenoidosa, which continued to increase until day 27. This aligns with studies on other Spirulina strains, where rapid growth is observed in the early stages of cultivation [⁵⁴,⁵⁵]. The slower but sustained increase in turbidity and cell count (cells/ml) in Chlorella is consistent with its prolonged exponential phase observed in earlier research [⁵⁶]. Biomass productivity (mg/L/day) was higher in S. subsalsa compared to C. pyrenoidosa during the experimental period, reflecting its faster growth rate. The protein content (mg/g FW) in Spirulina was highest around day 6, while Chlorella showed a peak in protein content around day 27. Pigment accumulation (mg/g FW) in both Spirulina and Chlorella followed distinct patterns. Total chlorophyll and carotenoid in Spirulina peaked on day 8 and day 12 respectively. This aligns with previous studies involving other strains of Spirulina (9). In contrast, C. pyrenoidosa showed maximum pigment content, including total chlorophyll and carotenoid on day 27. This delayed accumulation could be due to its extended growth phase, as reported in other Chlorella strains [⁵⁷]. As per the observations, S. subsalsa showed significantly higher growth and production of compounds like protein, chlorophyll and carotenoid when compared to C. pyrenoidosa. Thus, S. subsalsa can be considered as a more favorable source than C. pyrenoidosa for extraction of carotenoids. However, as S. subsalsa shows rapid growth, its requirement for medium constituents also becomes higher than C. pyrenoidosa.

Collectively, S. subsalsa is desirable for faster production of carotenoids favouring short-term cultivations, whereas, C. pyrenoidosa can be considered for slow and sustainable carotenoid productions in long-term cultivations. Experiments on optimization of growth parameters could result in increased carotenoid synthesis in C. pyrenoidosa. Upcoming experiments will explore extraction and identification of carotenoids from S. subsalsa and C. pyrenoidosa.

• Funding:
  This research received no external funding.

Acknowledgments:

None

Data availability statement:

Research data are available in the body of the manuscript.

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• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Jane Manfron

Publication Dates

• Publication in this collection
  06 Oct 2025
• Date of issue
  2025

History

• Received
  24 Nov 2024
• Accepted
  16 June 2025