Light Influences the Growth, Pigment Synthesis, Photosynthesis Capacity, and Antioxidant Activities in Scenedesmus falcatus

Light plays a significant role in microalgae cultivation, significantly influencing critical parameters, including biomass production, pigment content, and the accumulation of metabolic compounds. This study was intricately designed to optimize light intensities, explicitly targeting enhancing growth, pigmentation, and antioxidative properties in the green microalga, Scenedesmus falcatus (KU.B1). Additionally, the study delved into the photosynthetic efficiency in light responses of S. falcatus. The cultivation of S. falcatus was conducted in TRIS-acetate-phosphate medium (TAP medium) under different light intensities of 100, 500, and 1000 μmol photons m−2·s−1 within a photoperiodic cycle of 12 h of light and 12 h of dark. Results indicated a gradual increase in the growth of S. falcatus under high light conditions at 1000 μmol photons m−2·s−1, reaching a maximum optical density of 1.33 ± 0.03 and a total chlorophyll content of 22.67 ± 0.2 μg/ml at 120 h. Conversely, a slower growth rate was observed under low light at 100 μmol photons m−2·s−1. However, noteworthy reductions in the maximum quantum yield (Fv/Fm) and actual quantum yield (Y(II)) were observed under 1000 μmol photons m−2·s−1, reflecting a decline in algal photosynthetic efficiency. Interestingly, these changes under 1000 μmol photons m−2·s−1 were concurrent with a significant accumulation of a high amount of beta-carotene (919.83 ± 26.33 mg/g sample), lutein (34.56 ± 0.19 mg/g sample), and canthaxanthin (24.00 ± 0.38 mg/g sample) within algal cells. Nevertheless, it was noted that antioxidant activities and levels of total phenolic compounds (TPCs) decreased under high light at 1000 μmol photons m−2·s−1, with IC50 of DPPH assay recorded at 218.00 ± 4.24 and TPC at 230.83 ± 86.75 mg of GAE/g. The findings suggested that the elevated light intensity at 1000 μmol photons m−2·s−1 enhanced the growth and facilitated the accumulation of valuable carotenoid pigment in S. falcatus, presenting potential applications in the functional food and carotenoid industry.


Introduction
Light is one of the major factors that infuence algal growth, pigmentation, and photosynthesis, and light intensity can efectively regulate the metabolic induction of compounds that are valuable in responses to biochemical and physiological changes [1][2][3].Microalgae perform oxygenic photosynthesis, which converts light energy into chemical energy [4].Low light limits growth because the mutual shading of cells causes steep gradients of light, whereas high light accelerates photosynthetic electron transport, with the resultant production of reactive oxygen species (ROS) that can potentially damage cellular components, such as proteins, nucleic acids, and membrane lipids [2,5].
Microalgae are small unicellular photosynthesis organisms that live in saline or freshwater environments.Tey comprise diverse groups of microorganisms of some 72,500 species that use light and carbon dioxide for growth and biomass production [4,[6][7][8].Microalgae have been considered an important resource in biomass production since 1950 [9].Tey are great sources of value-added products that support the nutritional and energy needs of humans, such as proteins, lipids, vitamins, and antioxidants, and also are sources of pigments.Te production of biofuels using microalgae is expected to become a source of renewable energy in the future [4,8,10].
Te investigation into the antioxidant activity of microalgae has revealed their potential as a rich source of substances with high antioxidant capacity, positioning them as advantageous natural antioxidants.Microalgae exhibit abundance in pigments, phenols, polysaccharides, proteins, essential fatty acids, vitamins, and other bioactive nutrients of high value, thereby presenting a potent repository of natural antioxidants.Notably, while the prevailing market for natural antioxidants predominantly relies on land plants, antioxidants derived from microalgae are presently not widely available [11].Nevertheless, microalgae hold promise as a prospective source of natural antioxidant products due to their greater yield compared to terrestrial plants and the controllability of their culture conditions [12].In the realm of microalgae, phenolic and favonoid compounds have been identifed in numerous species, including Arthrospira maxima, Euglena cantabrica, Chlorella sp., Phormidium sp., Tetraselmis sp., Isochrysis sp., and Phaeodactylum sp.Several reports underscore the connection between phenolic acids and antioxidant concentrations within microalgae [13].Tis underscores the potential of microalgae as a prolifc source of diverse bioactive compounds and a promising avenue for developing natural antioxidants, ofering distinct advantages over their terrestrial counterparts.
One of the important groups of bioactive compounds produced by microalgae is carotenoids.More than 1100 carotenoids have been identifed in living organisms [14][15][16].Carotenoids are associated with photosynthesis in microalgae and serve to protect cells from oxidative damage and high light [15,17].Carotenoids also serve as commercially important nutrients in animal feed supplements, as colorants, and as compounds for cosmetic and pharmaceutical purposes [18].Of the several microalgae that produce lutein, Muriellopsis sp. and Scenedesmus almeriensis have been tested under growth conditions for large-scale biomass production [8].Te global carotenoid market had a value of approximately 1.5 million dollars in 2017 and reached 2 billion dollars by 2022 [15].
Scenedesmus, which belongs to the order Chlorococcales of the family Scenedesmaceae, is frequently dominant in freshwater lakes and rivers and is commonly found in fresh and brackish waters, particularly under nutrient-rich conditions [19].It was reported that Scenedesmaceae in Tailand comprised 35 taxa from 29 sampling sites [20].Many species of this genus are used worldwide for various purposes due to their ability to adapt to harsh environments and grow rapidly and because they are easy to cultivate and manage.Many researchers have reported on the efects of temperature, light, and pH on the growth of Scenedesmus spp.and on their uses that could improve pharmaceutical and nutraceutical properties for various applications [19,[21][22][23].In our previous study, it was shown that S. falcatus had high amounts of fatty acids, particularly in 28 compounds, and was a potentially good source of antioxidants with antidiabetic activities [24].Tis research sought to examine the impacts of varying light intensities on the growth, chlorophyll fuorescence, antioxidant characteristics, and augmentation of carotenoid content, with the overarching goal of efectiveness of utilizing S. falcatus (KU.B1). .Spectrophotometric determinations were performed using a UV-1800 UV-Visible spectrophotometer (Shimadzu Corp., Japan).Te chlorophyll fuorescence was determined by pulse amplitude modulation (PAM-2500, Walz GmbH, Germany), and the carotenoid analysis used the high-performance liquid chromatography (HPLC) alliance e2695 separation module (Waters Corp., USA) and column C 30 (YMC Co. Ltd., Japan).

Strains and Cultural Conditions.
Te strains of S. falcatus (KU.B1) were isolated and cultured in a liquid TRIS-acetate-phosphate (TAP) medium using the following method from a previous report [24].A temperature of 25 ± 1 °C and pH of 7.0 under diferent light intensities of 100, 500, and 1000 μmol photons m − 2 •s − 1 with a photoperiod of 12 : 12 were used.

Measurements of Growth.
Te optical density (OD) of 1 ml of the algal sample was measured at 750 nm (OD750) every 24 h up to 120 h using a UV-Visible spectrophotometer (UV-1800; Shimadzu Corporation, Japan) adapted from Chioccioli et al. [25].Te initial optical density of cell was set to 0.1 at 750 nm.
2.4.Measurements of Pigment.Te primary pigments in algae, namely, chlorophyll a, chlorophyll b, and total carotenoids, were analyzed using a UV-Visible spectrophotometer.Harvested cultures of 5 ml were obtained at intervals of 0, 24, 48, 72, and 120 h, followed by centrifugation at 15,000 rpm for 15 min at 25 °C.Subsequently, the pellet was subjected to extraction with DMSO, vortexed for 30 sec, and stored in the dark for 24 h.Te supernatant obtained was spectrophotometrically measured at 480, 649, 2 Scientifca and 665 nm wavelengths.Te calculation of pigment concentration was executed using the following equations [26]: ( Te pigment amounts were shown in micrograms per milliliter (μg•ml − 1 ).

2.5.
Measurements of Chlorophyll Fluorescence.Chlorophyll fuorescence assessments were conducted utilizing a pulse amplitude fuorometer (PAM-2500; Walz GmbH, Germany).Before measurements, algal cells were subjected to a dark acclimation period of 30 min.Subsequently, the efective quantum yield of photosystem II (YII) and the maximum quantum yield of photosystem II (Fv/Fm) were determined with an active light duration of 10 sec.Additional parameters were also quantifed, including non-photochemical quenching (NPQ), photochemical quenching (qP), and relative electron transport rate (ETR).

Preparation of Crude Extract.
A dried algal sample of 50 mg was soaked in acetone for 5 days at room temperature.Te crude extract was fltered through Whatman grade 1 flter paper and evaporated using a rotary evaporator at 40 °C with 250 mbar vacuum pressure and rotation at 120 rpm.Te crude extract was kept at − 20 °C in the dark until analysis was performed.

Antioxidant Activity.
Te DPPH scavenging capacity was demonstrated using a DPPH test, according to a previously described procedure [24].Separately, 150 μl of an extract of S. falcatus (KU.B1) was mixed with 150 μl of 0.2 mM DPPH solution in methanol (150 μl).After an incubation period of 30 min at 25 °C, the absorbance was measured at 520 nm.

Total Phenolic Compounds (TPCs).
Te Folin-Ciocâlteu test was used to measure the TPC of the algal extract.Tis test was performed according to the method developed by Khlif et al. [27] with some modifcations.A Folin-Ciocâlteu reagent solution was initially prepared by combining 2N Folin-Ciocâlteu reagent (10 ml) with distilled water (90 ml).For the sodium carbonate solution, 7.5 g of sodium carbonate (Na 2 CO 3 ) was dissolved in 100 ml of distilled water.For the assay, 25 μl of S. falcatus (KU.B1) extract was mixed with 125 μl of Folin-Ciocâlteu reagent solution.Te resulting mixture was allowed to stand at room temperature for 5 min.Subsequently, 100 μl of sodium carbonate solution was added, followed by thorough mixing, and the mixture was then incubated for 60 min.Te absorbance of the solution was measured at 765 nm.Te TPC content of the sample was quantifed in milligrams/grams of the gallic acid equivalent (GAE) by the calibration curve generated using the GAE concentration ranging from 0 to 1 mg•L − 1 : y � 0 � 0.2919x + 0.1298, R 2 � 0.8748.

Analysis of Carotenoids.
Te crude extract was redissolved in acetone, fltered through a membrane of 0.45 μm pore size, and injected in an amount of 20 μl into the HPLC e2695 separation module.For the mobile phase solvent, two mobile phases was used, A and B. Phase A was methanol-MTBE-water of 81 : 15 : 4, v/v/v, and phase B was methanol-MTBE-water of 7 : 90 : 4, v/v/v with a carotenoid C 30 column of 250 × 4.6 mm I.D. Detection was done with a PDA detector at a temperature of 25 °C and an arranged fow rate of 1 ml•min − 1 .Te UV wavelength scanning was 210 to 550 nm.

Statistical Analysis.
Te results were expressed as the mean of three replicated values, accompanied by the standard deviation (mean ± SD, n � 3).Statistical signifcance (p < 0.05) was assessed using both the analysis-of-variance (ANOVA) test and t-test in the context of a completely randomized design (CRD).

Efects of Light at Various Intensities on the Growth of S. falcatus.
Te investigation into the growth of S. falcatus (KU.B1) in TAP medium was conducted across varied light intensities of 100, 500, and 1000 μmol photons m − 2 •s − 1 .Te growth of S. falcatus (KU.B1) exhibited a discernible dependence on the applied light intensity.Notably, S. falcatus demonstrated exponential growth up to the 48 h (Figure 1(a)).Te maximum optical density values of the culture under 100, 500, and 1000 μmol photons m − 2 •s − 1 at 120 h were 0.78 ± 0.06, 1.15 ± 0.04, and 1.33 ± 0.03, respectively.After 24 h of cultivation, the cell culture exposed to light exhibited a gradual increase in intensity, appearing pale green (Figure 1(b)), eventually manifesting as a green culture after 120 h (Figure 1(c)), particularly notable in the cultivation under 1000 μmol photons m − 2 •s − 1 .

Chlorophyll Fluorescence.
In addition to its impact on algal cell growth and photosynthetic pigment levels, light intensity variations signifcantly infuenced photosynthetic efciency, particularly concerning photosystem II (PSII) activity.Chlorophyll fuorescence provides an informative tool to assess the efects of environmental stresses on plants, offering parameters such as Fv/Fm, Y(II), NPQ, qP, and ETR to quantify PSII efciency and assess photosynthetic performance [33].In our study, the response of S. falcatus (KU.B1) to varying light conditions revealed that high light intensity at 1000 μmol photons m − 2 •s − 1 led to the reduction in the Fv/Fm ratio and Y(II) value.Tis decline in photosynthetic efciency suggests a mechanism of inhibitory efects induced by high light stress.Te decrease in efciency may arise from either damage to PSII, where the photodamage rate surpasses the repair rate, or initiation of NPQ processes [34][35][36].Tis aligns with observations in other microalgae species, such as Dunaliella salina, which showed a reduction in the Fv/Fm ratio under higher light conditions exceeding 500 μmol photons m − 2 •s − 1 , indicative of photoinhibition [36].Similarly, in the case of Arthrospira platensis, a reduction in the Fv/Fm ratio during midday was attributed to intensifed solar radiation due to heightened light conditions [37].Furthermore, observations made on Alaria esculenta and Muriellopsis sp.Data are presented as mean ± standard deviation (SD) (n � 3).Diferent alphabets indicate signifcant diferences (a-c) within the same column at p < 0.05.
8 Scientifca indicated a decrease in the Fv/Fm ratio when exposed to high light conditions, consistent with our results [8,38].However, in our results, the photosynthesis continuously occurred under the high light intensity of 1000 μmol photons m − 2 •s − 1 .Tis intriguing observation implies that S. falcatus (KU.B1) may have developed an efcient repair cycle, enabling rapid turnover of damaged PSII components in response to heightened light intensity and maintaining optimal photosynthetic efciency [36].NPQ provides insights into the intra-thylakoid pH gradient and the chloroplasts' capacity to disperse excessive excitation energy as heat, which corresponds linearly to energy dissipation, efectively protecting cells from photodamage [36,39].Our investigation revealed that S. falcatus (KU.B1) cultivated under high light at 1000 μmol photons m − 2 •s − 1 exhibited minimal NPQ induction, suggesting that S. falcatus (KU.B1) may have efciently adapted to manage the intense light environment possibly due to the accumulation of substantial carotenoid quantities.Carotenoids contribute to NPQ by dissipating excess energy as heat [40].Te ability to control NPQ may help the alga balance light absorption and utilization for photosynthesis.In contrast with outcomes from a study involving Phaeodactylum tricornutum, exposure to high light at 280 μmol photons m − 2 •s − 1 resulted in a noticeable NPQ decline [41].
Te qP represents the proportion of light energy trapped by PSII reaction centers for electron transport [42].In our result, qP exhibited a slight decrease at the low light conditions (100 μmol photons m − 2 •s − 1 ) and high light conditions (1000 μmol photons m − 2 •s − 1 ) during a 24 h cultivation period.Previous studies on D. salina exposed to light conditions at 200, 500, and 1000 μmol photons m − 2 •s − 1 showed no fuctuations in qP, while a slight decrease was observed when cells were cultured under 1500 μmol photons m − 2 •s − 1 [36].
Te relative electron transport rate (ETR) stands as a pivotal parameter, ofering insights into the photosynthetic efciency of algae across varied light conditions [43,44].In our study, under high light conditions of 1000 μmol photons m − 2 •s − 1 , a signifcant reduction in ETR was observed in S. falcatus (KU.B1) after 120 h.Te decrease in ETR under high light conditions suggests that, despite the microalga thriving and exhibiting exponential growth under these intense luminous conditions, there is a limit to the photosynthetic capacity.High light intensities can lead to photoinhibition, a process where the rate of photodamage to the photosystem exceeds the repair rate, resulting in a decline in photosynthetic efciency [45].Te observed reduction in ETR may indicate a protective mechanism employed by S. falcatus (KU.B1) to mitigate the potential damage caused by excessive light.Microalgae often deploy various strategies to cope with high light stress, including thermal dissipation of excess energy (NPQ) and adjustments in the electron transport chain to optimize energy use [36,39].Te decrease in ETR might be a regulatory response to prevent overexcitation of the photosynthetic apparatus and to maintain a balance between energy absorption and utilization.Interestingly, the ability of S. falcatus (KU.B1) to exhibit sustained growth under high light conditions, despite the reduction in ETR, suggests that this microalga possesses adaptive mechanisms to acclimate to elevated light intensities efciently.S. falcatus (KU.B1) may have evolved into a dynamic photosynthetic apparatus, efectively managing and distributing absorbed light energy, even under stressful conditions.Tis observation also aligns with fndings from Chlamydopodium fusiforme, wherein a decreased ETR was noted during a four-day outdoor cultivation period [46].

Antioxidants and TPC.
Te DPPH assay evaluates the antioxidant capacity of tested compounds by measuring their ability to reduce DPPH radicals through direct electron transfer or radical quenching involving H atom transfer [47].Our results revealed a signifcantly elevated DPPH scavenging activity in S. falcatus (KU.B1) under optimal growth conditions with low and moderate light intensities (100 and 500 μmol photons m − 2 •s − 1 , respectively).Conversely, under high light intensity of 1000 μmol photons m − 2 •s − 1 , the DPPH scavenging activity was notably diminished.Elevated light intensity has the potential to induce oxidative stress in algae.Although algae produce antioxidants as a defense mechanism against oxidative stress, excessively high light conditions may surpass the antioxidant defense capacity, reducing scavenging activity [48].Excessive light can induce photoinhibition, a process where the photosynthetic apparatus is damaged, afecting the overall metabolic processes.Tis damage could decrease the production or efectiveness of antioxidant compounds, leading to reduced DPPH scavenging activity [49].Furthermore, high-light conditions might trigger a shift in the metabolic pathways of algae.Some pathways related to antioxidant production could be downregulated, or the energy resources could be redirected to cope with other stress responses, consequently impacting DPPH scavenging [50].Mishra et al. [51] reported enhanced growth along with increased amounts of carbohydrates, carotenoids, lipids, and antioxidative activity in Isochrysis galbana under high light at 325 μmol photons m − 2 •s − 1 .Furthermore, Dinev et al. [52] studied extracts from S. dimorphus and found that a high antioxidant potential determined by the DPPH method correlated with elevated levels of total phenolic and favonoid compounds.Increased phenolic production serves as a direct countermeasure against free radicals, interrupting the sequence of reactions in lipid peroxidation chain reactions [53].Consistently, our study demonstrated enhanced antioxidant efcacy from low light at 100 μmol photons m − 2 •s − 1 to high light at 500 μmol photons m − 2 •s − 1 , along with increased TPC content.

Individual Carotenoid Content.
Light intensity signifcantly infuences the quantity and composition of carotenoids, playing a pivotal role in the substantial accumulation of various carotenoids, including beta-carotene, lutein, and astaxanthin, within microalgae.Tis infuence demonstrates distinctive traits specifc to each species [17,54].Our results revealed high light-stimulated carotenoid synthesis in S. falcatus (KU.B1), dominantly with beta-carotene, lutein, and canthaxanthin, respectively.Carotenoids are essential pigments involved in photosynthesis.Tey capture light Scientifca energy and transfer it to chlorophyll, enhancing light absorption efciency and utilization in the photosynthetic process [55].One of the primary functions of carotenoids is to protect the photosynthetic apparatus from damage caused by excess light energy.Tey act as antioxidants, scavenging reactive oxygen species generated during photosynthesis and preventing oxidative stress [45].Algae often adjust the composition and concentration of carotenoids in response to changes in light conditions.Under high light intensity, algae may increase carotenoid production to enhance photoprotection, while they may reduce carotenoid synthesis under low light conditions.Tis is particularly important during stressful conditions, such as exposure to high light or environmental fuctuations.Under elevated light conditions, Scenedesmus sp.KGU-Y002 accumulated total carotenoids at 34.2 ± 3.8 mg•g − 1 dry weight per cell [56].In our study, beta-carotene consistently manifested as the preeminent carotenoid across all examined light intensities, followed by lutein.Earlier studies have outlined prevalent carotenoids in green algae, including beta-carotene, lutein, violaxanthin, and zeaxanthin.Notably, these carotenoids demonstrate a more extensive distribution among green algal species than higher plants [57].Lutein and betacarotene are classifed as primary carotenoids and play crucial roles in photosynthesis within the chloroplast [58].Beta-carotene, a primary microalgal carotenoid that transfers light energy to chlorophylls and mitigates oxidative damage in microalgae [16], accumulates in D. salina in response to increased light exposure [59].Microalgae, under suitable culture conditions, can accumulate lutein.For instance, under high light, microalgae utilize lutein to reduce oxidative damage through NPQ.Scenedesmus sp.accumulated approximately 7.47 mg/g dry weight or 19.70 mg/l/day of lutein when cultivated under 400 μmol photons m − 2 •s − 1 [60].Canthaxanthin, a secondary carotenoid, also shows light intensity-dependent accumulation [61].S. obliquus, a green alga, accumulates signifcant canthaxanthin [62].In the green alga Coelastrella striolata var.multistriata, the content of canthaxanthin, among a mixture of carotenoids, reached 47.5 mg/g dry weight at its maximum, depending on the light intensity [63].Exploring various species, Coulombier et al. [54] reported that Nephroselmis sp., Dunaliella sp., Picochlorum sp., Nitzschia sp.A, and Entomoneis punctulata exhibit elevated total carotenoid and individual carotenoid contents under high light conditions (600 μmol photons m − 2 •s − 1 ).Conversely, Tetraselmis sp., Schizochlamydella sp., Nitzschia sp.B, Talassiosira weissfogii, Cylindrotheca closterium, and Chaetoceros sp.demonstrate heightened total carotenoid content under a low light intensity (250 μmol photons m − 2 •s − 1 ).Te discernible impact of light intensity appears to be distinctly species-specifc.

Conclusions
Our investigation underscores the profound infuence of light intensity on the cultivation dynamics of Scenedesmus falcatus (KU.B1), infuencing key parameters such as biomass production, pigment content, and metabolic compound accumulation.Tis comprehensive study aimed to optimize light conditions, specifcally targeting the enhancement of growth, pigmentation, and antioxidative properties in S. falcatus (KU.B1).Cultivation under varying light intensities of 100, 500, and 1000 μmol photons m − 2 •s − 1 revealed a notable increase in S. falcatus (KU.B1) growth under high light conditions at 1000 μmol photons m − 2 •s − 1 , reaching a maximum optical density of 1.33 ± 0.03 and a total chlorophyll content of 22.67 ± 0.2 μg/ml at 120 h.Conversely, a slower growth was observed under low light at 100 μmol photons m − 2 •s − 1 .However, the enhanced growth under 1000 μmol photons m − 2 •s − 1 was accompanied by reductions in maximum quantum yield (Fv/Fm) and actual quantum yield (Y(II)), indicating a decline in photosynthetic efciency.Notably, the high light conditions at 1000 μmol photons m − 2 •s − 1 led to a signifcant accumulation of beta-carotene, lutein, and canthaxanthin within algal cells, showcasing the potential for valuable carotenoid pigment production.Tis suggests promising applications in the functional food and carotenoid industries.However, despite the positive impact on pigmentation and growth, antioxidant activities and total phenolic compounds decreased under high light conditions at 1000 μmol photons m − 2 •s − 1 .Te observed trade-of between enhanced growth and decreased antioxidative properties under high light conditions underscores the need for a balanced approach in optimizing light parameters for microalgae cultivation.Future studies could delve deeper into the intricate mechanisms governing this trade-of and explore strategies to mitigate potential oxidative stress while maximizing pigment production.Overall, these fndings contribute valuable insights into optimizing light conditions for microalgae cultivation, emphasizing the potential for S. falcatus (KU.B1) in applications related to biomass production and the carotenoid industry.
[29]ta are presented as mean ± standard deviation (SD) (n � 3).Diferent alphabets indicate signifcant diferences (a-b) within the same column at p < 0.05.− 2 •s − 1 , as evidenced by sustained exponential growth and elevated levels of photosynthetic pigments.Particularly noteworthy was the heightened growth and pigment accumulation observed in S. falcatus (KU.B1) under high light intensities at 1000 μmol photons m − 2 •s − 1 compared to 100 or 500 μmol photons m − 2 •s − 1 .Photosynthesis is a lightdependent process, and an optimal balance of light is crucial for energy capture and conversion into biomass[29].termediate chlorophyll synthesis between low and high light conditions.Te slower growth observed under low light conditions (100 μmol photons m − 2 •s − 1 ) correlates with a decreased chlorophyll content.S. falcatus (KU.B1) appears light-limited under these conditions, leading to a suboptimal photosynthetic rate and overall growth inhibition.Te