Biochemical profile of Scenedesmus isolates, with a main focus on the fatty acid profile

Abstract Biochemical characterization of new microalgal strains that are isolated from diverse environmental conditions is an important starting point for the establishment of high‐quality feedstock for nutraceutical and pharmaceutical applications. In this research study, the biochemical composition of three Iranian native subspecies of Scenedesmus microalgae (Scenedesmus obliquus, Scenedesmus bijugusi, and Scenedesmus sp.), with the main focus on fatty acid composition, was studied. The results showed that the strain Scenedesmus bijugusi had the highest biomass productivity (48 g/L/d), biomass (0.73%), carbohydrate (13.97%), fat (16.27%), protein (44.04%), chlorophyll‐a (6.32 mg/g), and carotenoids (3.7 mg/g). The lipid profile also revealed that S. obliquus had the highest percentage of polyunsaturated fatty acid (46.52%), ratio of ∑n‐3/∑n‐6 (5.96), ratio of polyunsaturated fatty acid to saturated fatty acid (PUFA/SAF) (1.18), α‐linolenic acid (22.74%), hypocholesterolemia index (1.61), and low atherogenic index (0.34). S. bijugusi and S. obliquus, thus, showed a great promise in nutraceutical and pharmaceutical applications due to their appropriate high productivity, biopigment, protein, lipid, antioxidant activity, long‐chain polyunsaturated fatty acids, and α‐linolenic acid.

iodine strains with a high potential to produce bioactive compounds from microalga (Dinpazhooh et al., 2022).
Essential fatty acids play a role in coronary heart diseases and strokes; they also play a role in cholesterol metabolism, cancer, blood pressure regulation, and diabetes improvement (Das, 2008).Marine microalgae such as Scenedesmus sp., Chlorella sp., Protothecoides, and Nannochloropsis Dunaliella salina have been used for the production of unsaturated fatty acids, such as omega-3 and α-linolenic acid (Ötleş & Pire, 2001;Sharma et al., 2015).
Bioprospecting of microalgae is defined as "the identification of economically valuable biochemical resources from algae rich in such content and enables industrial bioresource generation" (Khosravinia et al., 2023).Exploration of native microalgae and evaluation of the competency of bioactive metabolites and their productivity can be, therefore, highly significant.Moreover, native microalgae species of all countries are generally adapted to a wide range of their environments.Therefore, it is important to discover appropriate microalgae species with high biomass and special metabolites like Dunaliella sp. for nutraceutical and pharmaceutical applications (Araj-Shirvani et al., 2024;Gharajeh et al., 2020).Different varieties of isolated microalgae strains were considered for high-saturated fatty acids for biofuel production (Piligaev et al., 2015) or for pharmaceutical and biotechnological applications (Dinpazhooh et al., 2022).
Owing to the wide variety of natural resources and climate diversity, Iran has been considered as an excellent source for a large number of unstudied microalgal strains with special metabolic abilities.On the other hand, there has been a change of focus among researchers aiming to meet food needs from agriculture to investigate microalgae from different regions, given Iran's climate, vast area of land, and low rainfall.The present research was, therefore, conducted to find out more about the biochemical composition of Scenedesmus sp. as a microalga that is important due to its satisfactory lipid content for direct use or the use of its metabolites in the food and pharmaceutical industry.In this research, the biochemical composition of three native subspecies of Scenedesmus sp.
(Scenedesmus obliquus.IBRC-M-50130, Scenedesmus bijugusi.IBRC-M-50116, and Scenedesmus sp.IBRC-M50098), which were isolated from three different regions in Iran (Kharak, Hable Rood River of Garmsar, and Caspian Sea) was investigated.The biochemical compositions of three Scenedesmus sp. were described to achieve the main goal of the biotechnological and food industries: producing an economically advantageous nutrient as a substitute for agricultural products in dire situations such as droughts with low environmental damage.1).

| Biomass production
The measurement of the dry biomass weight was carried out based on the method proposed by Zhang et al. (2015).The microalgae inoculation (250 mL) was filtered on filter paper (0.45 μm) and then dried in an oven (60°C) for 12 h; finally, it was weighed (Zhang et al., 2015).
where A 662 , A 645 , and A 470 are absorbance wavelengths using the spectrophotometer (UV 2100, China) at 662, 645, and 470 nm, respectively.The obtained results were expressed as mg/g.

| Carbohydrate content
The measurement of total carbohydrates was done according to Bertrand's method (Heinze & Murneek, 1940).

| Preparation of the cell extracts for total phenolic
Cells were harvested by centrifugation (Universal 320R, Germany) at 3600 g for 20 min at 4°C, frozen at −80°C overnight, and freezedried (Dana Vacuum Industries, Iran).The freeze-dried biomass (0.2 g) was then thoroughly mixed in 5 mL of ethanol/water (3:1 v/v).
The tubes were sonicated in an ultrasonic water bath (Parsonic Ultrasonic, Iran) and shaken for 1 h at room temperature.The extracts were centrifuged, and the supernatant was collected (Bulut et al., 2019).
Then saturated sodium carbonate (2 mL) was added to this mixture.This mixture was obtained after shaking and further centrifuged (Universal 320R, Germany) at 11180 g for 10 min; it was allowed to stand for 30 min in the dark and the absorbance was measured at 750 nm by using the UV-vis spectrophotometer (UV 2100, China).
The calibration curve (Y = 0.0132x + 0.0968, R 2 = .98) of gallic acid was used to calculate TPC.The obtained results were then expressed as gallic acid equivalent (GAE) mg/100 g of the dry weight (DW) of the powder.

| Lipid extraction and GC analysis
Fatty acid and lipid analysis was determined by heating the 0.1 g lipid content of the microalgae samples at 80°C using 1 mL of sulfuric acid (2.5%) in methanol (98%) (1:40) for 90 min in screw-capped tubes.After the addition of 1.5 mL of 0.9% NaCl solution and 0.5 mL of hexane, fatty acids were extracted into the organic phase by shaking; the tubes were centrifuged at a low speed.The samples of the organic phase were separated through gas chromatography (Agilent 6890N Model USA) by applying a flame-ionization detector (FID) and a temperature program, including an initial temperature of 20°C, which was an increase of 50°C min −1 up to 250°C, by using an HP-88 (Agilent Technologies, USA, 100 m × 0.250 mm × 0.20) column.Helium was used as a carrier gas with a flow rate of 1.2 mL/min and a 1 μL injection volume with a split ratio of 10:1.Each peak was matched with the peak obtained by running the C4:C24 mix (Miquel & Browse, 1992).

| Nutritional and health value
Based on the fatty acid composition, the lipid nutritional quality was determined for the isolated Scenedesmus.Additionally, the atherogenic index (AI), thrombogenic index (TI), and hypocholesterolemic index (HI) were calculated using the equations ( 4)-( 6) (Gharajeh et al., 2020).
where AI is the atherogenic index, TI refers to the thrombogenic index, and HI indicates the hypocholesterolemic index.

| Statistical analysis
Statistical comparisons of the samples were performed using the one-way analysis of variance (ANOVA).Differences between the means were considered significant at the level of 5% (p < .05).The data were expressed as means ± standard deviations (SD) of three replicate determinations.

| Biomass dry weight and productivity
The biomass dry weight and productivity of Scenedesmus obliquus (0.64 g/L and 42 mg/L/day), Scenedesmus bijugusi (0.73 g/L, 48 mg/L/ day), and Scenedesmus sp.(0.55 g/L, 36 mg/L/day) are shown in Table 1.The biomass dry weight in our study was approximately similar to that previously recorded for Scenedesmus sp. and Chlorella sp.(about 1 g/L) (Visca et al., 2017), Chlorella (0.015 mg/mL) (Sharma et al., 2015); meanwhile, it was lower than that of Scenedesmus spp (4 mg/mL), Botryococus (4.5 mg/L) (Sharma et al., 2015), and Dunalilla sp.(2.6 mg /L) (Safi et al., 2014).The biomass productivity of Scenedesmus obliquus and Senedesmus abundans (Piligaev et al., 2015) was also similar to that found in this study.Of course, it should be noted that all 3 Scenedesmus isolated in Erlenmeyer grew in non-aerated and autotrophic conditions.

| Biochemical composition of the isolates
Protein, carbohydrate, lipid, ash, pigment (chlorophyll-a, chlorophyllb, and carotenoid), total phenol content, and antioxidant activity of the three isolated Scenedesmus were measured, as shown in Tables 1   and 2.

| Carbohydrate content
The carbohydrate content of S. bijugusi was 13.97%, which was higher than that of Scenedesmus sp.(9.35%) and S. obliguus (11.27%).The carbohydrate content of the 3 isolates was lower than that of Chlamydomonas reinhardtii (17%) (Chen et al., 2013), while it was higher than that of Dunaliella bioculata (4%) (Mäkinen et al., 2017) (Table 1).The results, thus, showed that S. bijugusi could be a suitable choice for the use of carbohydrates.The carbohydrate of microalgae is, therefore, a good candidate as an alternative to conventional sugars in the food fermentation process (Gharajeh et al., 2020).

| Ash content
The amount of ash in Scenedesmus sp.(20.2%) was significantly higher than that in the others (Table 1).The lowest amount of ash (14.9%) was observed in S. bijugusi.The amount of ash in Scenedesmus obliguus was 36.2% (Safi et al., 2014).

| Pigment content
Chlorophyll-a is the most abundant form of chlorophyll.In photosynthetic organisms, chlorophyll-a is the primary light-harvesting complex, in contrast to chlorophyll-b, which is an auxiliary lightharvesting pigment (Ünlü et al., 2014).The highest amount of chlorophyll-a was related to S. bijugusi (6.3 mg/g); meanwhile, the highest amount of chlorophyll-b belonged to S. obliguus (4.5 mg/g) (Table 2).C. vulgaris can potentially produce chlorophyll-a and b at 0.25-9.63 and 0.72-5.77mg/g, respectively (Safi et al., 2014).Scenedesmus of Iran was relatively suitable compared to Dunaliella sp.(Table 2).
Carotenoids are part of the photosynthetic apparatus, mainly in the reaction centers of photosystems, where they act as auxiliary pigments for light harvesting processes during photosynthesis and as structural stabilizers for building proteins; these compounds show high antioxidant activity (Swapnil et al., (2021).Carotenoid in S. bijuguusi was significantly higher (3.7 mg/g) in comparison to the others (Table 2).Carotenoids content in Dunaliella 3.5 mg/g (Gharajeh et al., 2020), 6.08-7.4 mg/g (Araj-Shirvani et al., 2024) C. vulgaris (0.4% DW) (Ru et al., 2020) and Scenedesmus sp.(0.15-0.80 mg/g) (Bulut et al., 2019) has been reported.It was observed that the carotenoids content of the three isolates of Scenedesmus was appropriate.
However, Scenedesmus sp. is not a good resource for the extraction of carotenoid (Table 2).

| Total phenolic content
Microalgae contain different types of phenolic compounds, such as vanillin, tannic acid, catechin, salicylic acid, ellagic acid, curcumin, quercetin, and benzoic acid.They are a group of bioactive compounds known for their antioxidant, anti-inflammatory, antimicrobial, anti-hypertensive, anti-arthritis, and heart protection activities (Bulut et al., 2019).In this research, no significant difference was observed between the three isolates of Scenedesmus sp. ( 6.5-7.7 mg GAE/g) (Table 2) (p > .05).The results obtained in this study were, thus, in good agreement with the phenolic concentration reported by Bulut et al. (2019).

| Antioxidant activity
The antioxidant capacity of the three isolates of Scenedesmus was evaluated in terms of DPPH radical scavenging capacity.The highest antioxidant activity was recorded for Scenedesmus obliquus (68.44%), while the lowest one belonged to S. bijugusi (54.55%) (Table 2).The antioxidant activity of Scenedesmus sp.25.65% was reported as well (Bulut et al., 2019), which showed that the 3 isolates of Scenedesmus had a more suitable percentage of the DPPH radical scavenging capacity.
The ratio ∑n-3/∑n-6 of S. obliquus and S. bijugusi is 5.96 and 1.94, respectively (Table 3).Consumption of oily foods is important in a healthy human life, provided that ∑n-3/∑n 6 fatty acids are balanced; this ratio is considered 5:1 by the World Health Organization (Gharajeh et al., 2020;Rubio-Rodríguez et al., 2010).The increase in fried foods and fast foods disrupts the balance of this ratio, causing a decrease in ∑n-3 in foods (Shanab et al., 2018).So, to maintain this balance, increasing the consumption of α-linolenic acid by enriching food products has been suggested (Silva et al., 2021).
Scenedesmus has a higher PUFA content, especially as a source of α-linolenic acid, as compared to other species; Arthrospira species, Chlorella, and Dunaliella (Custódio et al., 2014).Scenedesmus is also a rich source of unsaturated fatty acids (Ötleş & Pire, 2001), and S. bijugusi and Scenedesmus sp. are rich sources of α-linolenic acid (Table 3).

| CON CLUS ION
The determination of biochemical and bioactive components of the three isolated Senedesmus (especially Scenedesmus bijugusi. IBRC-M-50116 and Scenedesmus obliquus IBRC-M-50130) of Iran supported their potential nutraceutical and pharmaceutical applications due to medium-to high-protein and high-quality fats rich in PUFA and α-linolenic acids, appropriate health indices (high HI and low TI and AI), antioxidant activity, phenolic acid, and biopigment (Chlorophyll).However, it should be pointed out that all 3 isolated Scenedesmus were grown in Erlenmeyer under nonaerated and autotrophic conditions.Undoubtedly, biomass dry weight, protein, lipid content, PUFA, α-linolenic acids, appropriate health indices (high HI and low TI and AI), antioxidant activity, and phenolic acid of the three isolates would be increased by exposure under an optimized culture condition.Optimizing the growth conditions of these species in order to increase the accumulation of biomass and natural bioactive compounds and using them as whole or cracked cells to enrich food or produce new food products, along with technical and economic evaluations, can be recommended for future studies.
TA B L E 1Note: In each column, the averages with different letters are significantly different at the five percent level of the LSD test.
Fatty acid profile (%) of isolated Scenedesmus obliquus IBRC-M-50130, Scenedesmus bijugusi IBRC-M-50116, Scenedesmus sp.IBRC-M50098, and other green microalgae.In each row, the averages with different letters are significantly different at the five percent level of the LSD test.
Leptocylindrusdanicus), as shown in Table4.The lowest AI and the highest HI, which could be an indicator of atherogenic health, play a role in reducing cardiovascular diseases.AI and TI are two important indicators of nutritional health that show platelet aggregation ability.Foods with low AI and TI levels (containing less saturated fatty acids) have a greater ability to protect against such diseases.AI in the oil of Scenedesmus sp., S.obliquus, and S. bijugusi was 0.55, 0.34, and 0.41, respectively, which was partly equal to that of Dunaliella sp.ABRIINW-G2/1 (0.39) (Table4).TI and HI values in the three isolates of Scenedesmus were approximately comparable with those of Dunaliella sp.