Physiological resilience of freshwater microalgae Chlorella sorokiniana under NaCl stress supplemented with sodium sulfate as a mitigating agent

Physiological resilience of freshwater microalgae Chlorella sorokiniana under NaCl stress supplemented with sodium sulfate as a mitigating agent | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Physiological resilience of freshwater microalgae Chlorella sorokiniana under NaCl stress supplemented with sodium sulfate as a mitigating agent Ankush Yadav, Laishram Amarjit Singh, Suhani Sharma, Rupesh Bhardwaj, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7804893/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Microalgae show remarkable resistance to abiotic stresses such as salt. This work investigates the effects of sodium sulfate (Na 2 SO 4 ) and sodium chloride (NaCl) on the freshwater microalgal strain Chlorella sorokiniana with particular emphasis on physiological activities. The study is focused on to understand the molecular processes of C. sorokiniana in a controlled environment using NaCl and equimolar concentrations of NaCl and Na 2 SO 4 . Certain biochemical assays were resulted C. sorokiniana , cultured with a salt mixture of 350 mM (NaCl + Na 2 SO 4 ), showed better growth than 350 mM NaCl. The ROS production was high in 350 mM NaCl, which is 1.71 and 1.95 times higher compared to the control and equimolar concentration of NaCl + Na 2 SO 4, respectively. The osmolyte level in 350 mM NaCl increased by 2.9 and 2.29-fold as compared to the control and equimolar concentration of NaCl + Na 2 SO 4 , respectively. Significant alterations in protein expression suggested that stress-response pathways have been activated. Furthermore, significant fluctuations in omolyte and antioxidant levels indicate the significant adaptation of microalga to salinity stress. These findings contribute to the optimization of microalgae farming by shedding light on the physiological and biochemical strategies of microalgae in saline environments and essential to comprehend in order to advance biotechnological applications that are sustainable. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Plant sciences Microalgae Chlorella sorokiniana NaCl Na2SO4 Isozymes Antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Microbes play an essential biogeochemical role in the ecosystem. Microalgae are attracting study interest because of their capacity to produce diverse substances, rapidly increase biomass, and alter their biochemical composition in response to changing growth circumstances [ 1 , 2 , 3 , 4 , 5 ]. Microalgae, due to their ability to efficiently use carbon dioxide and water, make them sustainable in the soil where traditional crops are unsuitable [ 6 , 7 ]. Salinity has a substantial effect on the development and productivity of microbial communities such as microalgae, fungi, and bacteria. It changes ionic balance and osmotic potential, affecting photosynthesis, respiration, and nutrient intake [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Microalgae, having a remarkable tolerance to different abiotic stresses, employ various physiological and biochemical strategies to thrive and prosper in a saline environment [ 17 , 18 , 19 , 20 , 21 ]. Microalgae in stress conditions have been found to produce more lipid content. Some microalgae, such as Desmodesmus abundans and Chlorella pyrenoidosa , have been found to produce higher lipid content when exposed to certain concentrations of salt stress [ 22 , 23 , 24 , 25 ]. Different concentrations of salt affect growth, biomass yield and lipid accumulation of Chlorella vulgaris and Scenedesmus obliquus [ 26 , 27 ]. Sodium chloride (NaCl) and Sodium sulfate (Na 2 SO 4 ) are often used to adjust salinity levels. NaCl being cost-effective and user-friendly, is often used to induce lipid content in microalgae [ 28 ]. However, the high lipid content produced by this procedure affects the biomass quantity and thus overall lipid production. Therefore, it is necessary to overcome this contradiction and develop cultures that allow optimal growth while also producing high lipid content. Supplementing with Na 2 SO 4 has been reported to increase the production of biomass and promote growth in species such as Chlamydomonas moewusii and Spirulina platensis [ 29 , 30 ], Scenedesmus quadricauda [ 31 ], and Chlorella pyrenoidosa [ 32 ]. The synergistic effects of NaCl and Na 2 SO 4 on the development and productivity of microalgae remain little understood. This research aims to examine the impact of individual salt (NaCl) and combination salt (NaCl + Na 2 SO 4 ) on protein expression, osmolyte levels, and antioxidant concentration levels in microalgae. 2. Results 2.1. Isolation and Characterization of Chlorella sorokiniana Morphological observation under a light microscope revealed characteristic spherical to oval-shaped cells typical of Chlorella species (Supplementary Fig. 1). The result of PCR showed an amplified gene fragment length of approximately 1800 bp (Supplementary Fig. 2). The resulting sequence was subjected to BLAST analysis using the NCBI database, which confirmed the strain as Chlorella sorokiniana , which is highly homologous to Chlorella sorokiniana isolate NS5 (KM502980.1) with 99.09% sequence similarity. The sequence was subsequently submitted to the NCBI GenBank and received accession number PV648655. The phylogenetic tree was constructed and confirmed the organism as Chlorella sorokiniana (Supplementary Fig. 3). 2.2. Estimation of Growth and Chlorophyll Content The microalgae were grown in BG-11 medium with equimolar concentrations of 350 mM (NaCl) and 350 mM (NaCl + Na 2 SO 4 ), along with a control condition. Growth analysis showed better growth in the culture treated with 350 mM (NaCl + Na 2 SO 4 ) as compared to the culture treated with 350 mM (NaCl), as shown in Fig. 1 . The specific growth rate (µ), division rate per day (Dd), and doubling time (tD) all followed a consistent trend, showing a 2.74-fold decline in µ and Dd and a 2.74-fold increase in tD under 350 mM NaCl treatment, while the combined treatment with 350 mM NaCl + Na₂SO₄ exhibited a lesser impact, with a 1.75-fold reduction in µ and Dd and a 1.75-fold increase in tD compared to the control. In terms of biomass productivity, a significant 4.86-fold decrease was observed under NaCl stress, whereas the combined salt treatment resulted in a comparatively lower 2.54-fold reduction, indicating partial mitigation of salt-induced growth inhibition by sodium sulfate supplementation (Table 1 ). Chlorophyll yield was estimated on the 8th day, which showed the high chlorophyll yield in the control, followed by salt mixture 350 mM (NaCl + Na 2 SO 4 ), and last 350 mM (NaCl) as shown in Fig. 2 (A). Chlorophyll yield per mg of dry cell weight was also estimated. The culture treated with 350 mM NaCl yielded approximately only 27% compared to the control, while the culture exposed to combined salt treatment retained approximately 56% of the chlorophyll yield relative to the control, as shown in Fig. 2 (B). While both salts induced stresses leading to a decrease in chlorophyll yield, the combination of NaCl and Na 2 SO 4 showed better chlorophyll than that of the culture with only NaCl. The presence of SO 4 ‾ may possibly lessen the salt toxicity. Table 1 Growth Kinetics of Chlorella sorokiniana Under 350 mM NaCl and 350 mM (NaCl + Na₂SO₄) Treatments Sr. No. Treatment Specific Growth Rate (µ) (day⁻¹) Division per Day (Dd) (div·day⁻¹) Doubling Time (tD) (days) Biomass Productivity (cells·mL⁻¹·day⁻¹) 1. Control 0.210 0.303 3.30 4.119 × 10⁶ 2. 350 mM NaCl 0.0766 0.1105 9.05 8.473 × 10⁵ 3. 350 mM (NaCl + Na₂SO₄) 0.120 0.1732 5.77 1.6219 × 10⁶ 2.3. Sucrose Estimation Sucrose content was quantified to evaluate osmolyte accumulation in Chlorella sorokiniana (PV648655) cultures exposed to varying concentrations of salt stress. Measurements were conducted on alternate days using a sucrose standard curve for calibration. The level of cellular sucrose production significantly increased with an increase in salt exposure time and attained its maximum on the 8th and 10th days of salt exposure. The increment in sucrose production was 2.9 and 2.29 folds noted in cells exposed to 350 mM NaCl and 1.6 and 1.61 folds in cells exposed to a salt mixture of 350 mM (Na 2 SO 4 + NaCl) in comparison to the control (Fig. 3 ). 2.4. Antioxidant Enzyme Activity and DPPH Radical Scavenging Assay The antioxidant activity in C. sorokiniana (PV648655) cells exposed to different concentrations of salt stress was assessed using the DPPH radical scavenging assay and superoxide dismutase (SOD) activity measurement. After 72 hours of salt stress, DPPH radical scavenging activity and SOD activity were found to be 1.71- and 1.95-fold higher, respectively, in cells treated with 350 mM NaCl compared to the control. In cells treated with a 350 mM salt mixture (NaCl + Na₂SO₄), these values increased by 1.38- and 1.10-fold, respectively, compared to the control as shown in Fig. 4 . 2.5. Native PAGE of Superoxide Dismutase Isozyme profiling of antioxidative enzymes showed three isoforms (61.9, 56.5, and 51.9 kDa) of SOD in control and salt-stressed cells. The intensity of bands was high in cells with 350 mM NaCl and a salt mixture of 350 mM (NaCl + Na 2 SO 4 ). An additional band of isozyme was found in cells treated with NaCl of molecular weight between 61.9 and 60 kDa, as shown in Fig. 5 . Isozymes are enzymes that catalyze the same reaction but have a different molecular formula. 2.6. SDS PAGE The result of SDS PAGE showed that the protein bands upregulation was higher in equimolar concentration 350 mM NaCl-treated cells than in 1:1 mixture-treated cells with respect to the control. Downregulation of protein bands was high in the cells treated with a 1:1 salt mixture with respect to the control. The bands of molecular weight 168.5, 141.7, 101.8, 69.9, and 53.3 kDa were highly downregulated in the 1:1 salt mixture, respectively, to the control (Fig. 6 ). 2.7. Nile Red Assay Intracellular lipid accumulation was observed using Nile red dye. Accumulation of lipids was higher in the cells treated with 350 mM NaCl than in cells treated with the salt mixture (Fig. 7 ). Recent studies discussed under stress condition increament of ROS are closely related to total lipid content in microalgae but the exact mechanism behind this phenomenon remains to be explore [ 33 , 34 ]. The green images are due to chlorophyll and red colour cells showing autofluorescence. 2.8. ROS detection Reactive oxygen species (ROS) levels, indicating total cellular oxidative stress, were assessed using the H₂DCFDA dye, and the relative fluorescence intensity was recorded, as shown in Fig. 8 . Under 350 mM NaCl stress, fluorescence intensity increased by 1.63-fold compared to the control, while cells exposed to 350 mM (NaCl + Na₂SO₄) showed a 1.29-fold increase. Notably, ROS levels in the combined salt treatment were reduced by 1.26-fold relative to NaCl alone. These findings indicate that NaCl stress induces excessive ROS accumulation, leading to potential cytotoxicity. In contrast, the reduced ROS generation under the combined salt stress suggests a protective effect of sulfur, partially mitigating oxidative stress. This supports the role of sulfur supplementation in alleviating the harmful effects of salinity-induced oxidative damage in microalgae. 2.9. Cell Apoptosis detection by Annexin-V FITC Flow cytometry analysis showed that under control conditions, 96.3% of the cells (LL region) were viable, with 2.8% in the early apoptotic stage (LR region) and 0.4% in the late apoptotic stage (UR region). Under 350 mM NaCl stress, cell viability decreased to 91.6%, while early and late apoptotic cells increased to 6.7% and 1.0%, respectively (Fig. 9 ). In contrast, cells exposed to the combined salt stress (350 mM NaCl + Na 2 SO₄) showed slightly improved viability at 93.4%, with 4.9% early apoptotic and 0.8% late apoptotic cells. These results suggest that sulfur supplementation reduces salt-induced apoptosis and helps maintain higher cell viability. 3. Discussion Salinity suppresses the growth and yield of C. sorokiniana (PV648655); however, NaCl alone induced more toxicity in comparison to the equimolar concentration of the NaCl + Na 2 SO 4 mixture. In a similar experiment conducted in Anabaena fertilissima , it was found that oxidative stress is lower in those cells exposed to the salt mixture in comparison to those cells exposed to NaCl only [ 35 ]. In stressed conditions, antioxidant molecules such as glutathione, thioredoxin, and glutaredoxin are rapidly synthesized to mitigate stress and facilitate the normal development of microalgae [ 36 , 37 , 38 , 39 ]. The presence of SO 4 − in the mixture might reduce the stress as SO 4 − can be readily incorporated into protein as the redox-active cysteine residue and be used in synthesizing the antioxidants [ 40 , 41 ]. It has been found that sulfate in mustard increases glutathione production, which in turn increases salt tolerance [ 42 ] while its deprivation has been found to reduce glutathione content [ 43 ]. Chlorophyll yield can be used as an indicator of photosynthetic health in C. sorokiniana PV648655. High salinity leads to ionic and osmotic imbalances, disrupting photosystem and chlorophyll synthesis [ 44 , 45 , 46 , 47 ]. The negative effect of salt on microalgal proliferation was further confirmed by the growth kinetics data, indicating partial mitigation of salt toxicity. This highlights the role of sulfate in maintaining better growth dynamics even under high saline conditions, possibly by supporting key metabolic and stress-alleviation pathways. The chlorophyll yield under the combined salt condition (350 mM NaCl + Na 2 SO 4 ) was found to be higher than in the 350 mM NaCl treatment alone. It may be because SO 4 – might have mitigated some of the detrimental effects caused by high Na + . The presence of SO 4 – likely helps in maintaining cellular integrity under osmotic stress by contributing to the synthesis of osmoprotective molecules [ 41 ]. The algae employ a ROS-scavenging strategy to fight higher production of ROS, which includes enzymatic and non-enzymatic mechanisms [ 48 , 49 ]. Salinity increases the activity of antioxidants such as SOD, CAT, and GR to overcome oxidative stress and maintain cellular redox homeostasis at its optimum [ 50 , 51 , 52 ]. The antioxidative enzyme activity significantly increased in the cells exposed to 350 mM NaCl compared to 350 mM NaCl + Na 2 SO 4 exposed cells, suggesting C. sorokiniana experiences elevated levels of ROS under high salinity. The lower antioxidative enzyme activity in the equimolar mixture of NaCl and Na 2 SO 4 exposed cells suggests that the presence of SO 4 ˉ could help in reducing oxidative stress. It has been reported that sulphur is the precursor of the synthesis of sulfur-containing antioxidants such as glutathione and other thiol compounds, which are major cellular antioxidants and detoxify ROS to maintain the redox balance. Therefore, the presence of sulfate enhances the cell’s ability to cope with oxidative stress [ 35 , 41 ]. The uptake of ions by microalgal cells follows the specific membrane transport systems, located in the plasma membrane. Na⁺ enters the cell via Na⁺/H⁺ antiporters, which help to regulate ion homeostasis and osmotic balance under saline or stress conditions [ 53 ]. In case of Cl⁻, transport through anion channels and H⁺/Cl⁻ symporters, to maintain turgor pressure [ 54 ]. While SO₄²⁻ are taken up through the sulfate transporter. This helps to mediate sulfate uptake for assimilation into sulfur-containing amino acids like cysteine and methionine [ 55 ]. (Nagesh et al., 2024). The result of the experiment points to a potential protective mechanism where SO 4 ˉ supports cellular redox balance, thereby lessening the dependence on enzymatic antioxidants. Superoxide dismutase (SOD) isozyme profiling under conditions of salt stress demonstrates a multifaceted and adaptable antioxidative response in cells. An elevated oxidative stress response to NaCl is shown by the detection of three SOD isoforms (61.9 kDa, 56.5 kDa, and 51.9 kDa) in both salt-stressed and control cells, with greater band intensity in cells treated with 350 mM NaCl. The discovery of a second SOD isoform that is only present in cells treated with NaCl and ranges in size from 61.9 to 60 kDa points to the induction of a novel isozyme that is only responsive to stress caused by NaCl. These results highlight the functions of various SOD isozymes in oxidative stress management, including Cu/Zn-SOD in the cytosol and chloroplasts, Mn-SOD in mitochondria and Fe-SOD in the cytoplasm and chloroplasts [ 56 ]. The observed increase in total soluble protein content and altered banding pattern in SDS-PAGE under NaCl + Na₂SO₄ treatment suggests that the combined salt stress altered intracellular protein expression patterns, possibly through modulation of stress signaling and sulfur assimilation pathways. The presence of SO₄²⁻ may support enhanced synthesis of stress-related proteins by improving redox balance, sulfur-containing amino acid availability (e.g., cysteine, methionine), and antioxidant defense, rather than directly affecting protein solubility in the medium [ 57 , 58 ]. Therefore, the differences in protein band intensity are attributed to changes in cellular stress physiology, not to extracellular ionic effects on solubility. The addition of NaCl increases the ionic strength of the solution, leading to the "salting out" effect, where proteins become less soluble and may precipitate out of the solution [ 59 ]. This results in a lower observed protein concentration (1.576 µg/µl). When Na₂SO₄ is added along with NaCl, the solution contains both Na⁺, Cl⁻, and SO₄²⁻ ions. Sulfate ions affect enhancing protein solubility by stabilizing the hydration shell around proteins and reducing protein-protein interactions that lead to aggregation and precipitation [ 60 ]. This stabilization effect counteracts the salting-out effect of NaCl, leading to a higher observed protein concentration (3.882 µg/µl). The observed increase in protein concentration is due to electrostatic stabilization, hydration shell formation, and charge screening. NaCl alone promotes "salting out" and reduces protein solubility, while the addition of Na 2 SO 4 introduces sulfate ions that enhance protein solubility through various stabilizing interactions, leading to a net increase in the observed protein concentration in the NaCl + Na 2 SO 4 solution. Results observed through the Nile red assay showed that lipid accumulation was notably higher in cells treated with 350 mM NaCl compared to those in the combined salt treatment. The difference in intracellular lipid accumulation between cells treated with 350 mM NaCl alone and those treated with 350 mM NaCl + Na 2 SO 4 likely arises from the varying effects of these salts on cellular processes and metabolism. High concentrations of NaCl can induce osmotic stress, triggering various cellular responses, including changes in gene expression and metabolism. This stress can activate signaling pathways such as the MAPK and NF-κB pathways, which regulate cellular responses to stress, including inflammation and lipid metabolism. Additionally, osmotic stress can lead to increased lipogenesis, resulting in enhanced lipid accumulation within cells [ 61 , 62 ]. High salt concentrations can disrupt cellular homeostasis, including ion balance and energy metabolism, further contributing to changes in lipid metabolism and accumulation. Lipid bodies can sequester excess ROS and act as energy reservoirs during stress conditions. The high lipid accumulation in NaCl alone-treated cells suggests that microalgae channel resources toward lipid synthesis to cope with oxidative damage. The addition of Na 2 SO 4 introduces sulfate ions into the cellular environment, potentially altering cellular processes. The presence of sulfate ions alongside NaCl may moderate osmotic stress, reducing the extent of cellular responses associated with osmotic stress, including changes in lipid metabolism. The reduced apoptosis observed under combined salt treatment likely reflects the role of sulfur in reinforcing cellular defense and maintaining membrane integrity under stress conditions. Once sulfate ions are taken up by specific transporters they are assimilated into cysteine through a tightly regulated sulfur assimilation pathway. Cysteine serves as a precursor for glutathione and other thiol-containing antioxidants, which are pivotal in detoxifying ROS and stabilizing redox homeostasis [ 63 ]. By supporting the biosynthesis of such protective molecules, sulfur reduces cellular damage, prevents oxidative-triggered programmed cell death, and enhances overall stress resilience. This protective mechanism parallels the concept of sulfur-enhanced defense (SED) observed in higher plants [ 64 ], suggesting that in Chlorella sorokiniana , sulfur availability not only contributes to metabolic demands but also actively modulates cellular survival pathways under abiotic stress. 4. Conclusion Chlorella sorokiniana exhibited alterations in protein expression under salt stress conditions. However, the combined treatment of NaCl and Na₂SO₄ alleviated the adverse effects induced by NaCl alone. The presence of Na₂SO₄ appeared to mitigate NaCl-induced stress, as evidenced by a reduction in antioxidant activity compared to cultures treated solely with NaCl. The findings conclude that Na 2 SO 4 is a potent mitigating agent for salt (NaCl) stress in C. sorokiniana , this underscore the adaptive mechanisms of C. sorokiniana . Further, research should aim to clarify the molecular signalling pathways involved in the salt stress responses and explore the potential of combining different salts to boost the resilience and productivity of microalgae. 5. Materials and Methods 5.1. Isolation and Characterization of Chlorella sorokiniana Fresh water microalgal samples collected from the pond of Guru Nanak Dev Thermal Power Plant, Bhatinda, India. Samples were transferred to BG-11 media to obtain an axenic strain as described in the methods [65]. The isolated strain was observed under a light microscope (Nikon Eclipse Ci-L plus) using a 60× objective lens with bright-field contrast to examine its morphological features. Genomic DNA was isolated using the CTAB method [66] for molecular characterization and amplified by polymerase chain reaction (PCR) using primers targeting the 18S ribosomal RNA gene region. The primer sequences used were: forward (18S-F) 5′-GTCATATGCTTGTCTCAAAGATTAAGCC-3′ and reverse (18S-R) 5′-CCTTGTTACGACTTCTCCTTCCTCTAA-3′. The amplified product was analyzed using the NCBI BLAST (Basic Local Alignment Search Tool) for taxonomic identification and subsequently submitted to the NCBI database to obtain an accession number. 5.2. Growth Measurement 5.2.1. Growth Conditions Experiments were carried out in the 500 ml conical flask containing 300 ml BG-11 culture media to study the impact of NaCl, Na 2 SO 4 + NaCl salt [67]. First, the exponential phase culture (300 ml) of C. sorokiniana was centrifuged for 5 minutes at 7000 g at 25 o C and subsequently introduced into flasks containing equimolar concentrations of 350 mM NaCl, 350 mM (NaCl + Na 2 SO 4 ), along with the culture in the control condition. Further, the cultures were maintained in a culture chamber set at 28 ± 1 o C and under a 16:8 h light:dark photoperiod with a light intensity of 50 µmol/m²/s throughout the experiment. 5.2.2. Estimation of Growth and Chlorophyll Content 5.2.2.1. Optical Density-Based Growth Monitoring The growth of Chlorella sorokiniana was monitored daily by measuring optical density at 750 nm using a double-beam UV-Visible spectrophotometer (Motras Scientific, India). The culture reached an OD₇₅₀ of 1.0 on the 8th day of cultivation, which was used as the reference point for further analyses. 5.2.2.2. Growth Kinetics A standard curve of optical density (OD₇₅₀) versus cell count was generated using C. sorokiniana cultures at varying cell densities. Cell enumeration was performed using a hemocytometer (Improved Neubauer chamber) following the protocol described by Zhang (2020) [68]. To ensure data reliability, all cell counts were conducted in triplicate. Growth kinetics were evaluated by calculating the specific growth rate (μ), division rate per day (Dd), doubling time (td), and biomass productivity (P), as described by Gani et al., (2016) [69], using the following equations: Specific growth rate (µ/day) = ln (Nf/Ni)/ Tf-Ti Division per day (Dd) = µ/day / ln2 Doubling time (td) = 1/ Dd Biomass productivity (cell/mL/day) = (Nf/Ni)/ Tf-Ti Where Ni and Nf are the initial and final cell concentrations (cells·mL⁻¹), and Ti and Tf are the respective time points (in days). The exponential growth phase was estimated using a minimum of three time points, plotting the growth curves for parameter estimation accurately. 5.2.2.3. Chlorophyll Content Estimation Chlorophyll (chl) estimation was carried out at this point, following the procedure described by Kirk & Allen (1965) [70]. For the analysis, 5 ml of microalgal culture was centrifuged at 9000 × g for 10 minutes in a 15 ml centrifuge tube. The supernatant was discarded, and the pellet was resuspended in 5 ml of 80% acetone, thoroughly mixed by vortexing, and incubated overnight in the dark at 4°C. After centrifugation, chl µg/ml was calculated using a spectrophotometer to measure the absorbance at 663.6 nm and 646.6 nm against acetone as blank. The concentration of Chlorophyll a and Chlorophyll b were evaluated according to the following equations [71]. Chlorophyll a (µg chlorophyll/ml medium) = (12.25E 663.6 – 2.55E 646.6 ) / V Chlorophyll b (µg chlorophyll/ml medium) = (20.31E 646.6 – 4.91E 663.6 ) / V Chlorophyll a+b (µg chlorophyll/ml medium) = (17.76E 646.6 + 7.34E 663.6 ) / V Where: E 663.6 and E 646.6 represent absorbances at 663.6 nm and 646.6 nm minus absorbance at 750 nm, respectively V = Volume of the sample (ml) 5.3. Estimation of sucrose Estimation of sucrose was done using the method Van Handel (1968) [72]. 50 ml of microalgal culture were centrifuged at 7000 g at 25°C for 5 min. Pellets were homogenized in 80% ethanol to extract soluble sugars and kept in a water bath set at 100°C for 5–10 minutes. 100 µL of 30% KOH were added to the residue and again kept in a water bath at 100°C for 5 minutes. The samples were allowed to cool to room temperature, and then 3 ml of anthrone reagent was added and warmed up at 40°C for 15 minutes. The samples were allowed to cool down at room temperature, absorbency was recorded at 620 nm wavelength. Sucrose concentrations were calculated by comparing the absorbance values to a standard curve prepared with known concentrations of sucrose processed identically. 5.4. Determination of Antioxidative Enzyme 5.4.1. DPPH Assay Antioxidant activity was assayed by the method of Dawidowicz et al., (2012) [73], using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The standard was prepared for the DPPH assay against ascorbic acid of a known different concentration. A stock solution of 1 mM DPPH (methanol) and an ascorbic concentration (5–70 μg/ml) was prepared in methanol. The absorbance was recorded at 517 nm after 30 minutes of incubation in the dark on a microplate reader (Synergy H1, BIOTEK). 5.4.2. Quantification of Superoxide Dismutase Superoxide dismutase (SOD) activity was determined by measuring the inhibition of photochemical reduction of the nitro blue tetrazolium (NBT) method by Beauchamp & Fridovich (1971) [74]. In a test tube, a 1 ml reaction mixture containing phosphate buffer (50 mM pH -7.8), 9.9 mM methionine, 0.025% Triton-X 100, 57 μ mol/l NBT, and 20 μl extracted enzyme was added. After that, 10 μl of riboflavin (4.4 mg/100 ml) was added. The tubes were kept under 20-watt fluorescent bulbs. After 15 minutes, the absorbance was recorded at 560 nm. 5.4.3. Native PAGE In order to ascertain the expression of distinct antioxidant enzyme isozymes, an equal amount of protein extract was loaded onto 10% native polyacrylamide gel and electrophoresed at a constant voltage of 150 V at 4 o C till the dye reached the bottom of the gel method as described by Sambrook and Russell (2001) [75]. The image of the gel was captured by gel-doc after enzyme-specific staining of the gel. 5.5. Protein Analysis Protein was extracted in accordance with Hurkman and Tanaka (1986) [76]. The exponential growth of microalgae culture was centrifuged at 9000 × g for 15 min. The pellet was lyophilized and crushed into a powder using liquid nitrogen. Crushed cells were suspended in 2 ml of protein extraction buffer (0.7 M sucrose, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCL, 2 mM PMSF (phenylmethanesulfonyl fluoride), and 0.005% Triton-X). The mixture was collected in a microcentrifuge tube and centrifuged at 3040 g for 50 minutes at 4 o C. The supernatant containing soluble protein was collected in a separate tube and stored at -20 o C for further experiments. 5.5.1. SDS PAGE An equal concentration (50μg/μl) of extracted protein was loaded onto SDS PAGE 10% (Sambrook and Russell, 2001) [75] along with a protein marker (10- 245 kDa), and the gel was run at 100 V for the stacking gel and 150 V for the resolving gel. The gel was removed when the dye reached its bottom and stained with Coomassie brilliant blue (CBB) R-250. 5.6. Nile Red Assay Nile red assay was done by the method of Zhao et al. (2019) [77]. 1 ml of culture was taken and centrifuged at 1137 × g for 3 minutes. The supernatant was discarded, and the pellet was resuspended in 200 μl of 20% DMSO and kept for 2 hours at room temperature. The suspension was again centrifuged at 1137 x g for 3 minutes, and a pellet was taken and resuspended in water. The prepared stock solution of Nile Red (1 mg/ml of absolute acetone). Nile red (5μl) was added to the algal cells and incubated for 10 minutes in the dark. The image of algal cells was taken at 530 nm excitation and 575 nm emission wavelengths. 5.7. Determination of cytotoxicity 5.7.1. ROS determination Intracellular ROS detection was done using 2’7’-dichlorodihydrofluorescein diacetate (H 2 DCFDA) dye [78]. The non-fluorescent dye H 2 DCFDA is oxidized by intracellular reactive oxygen species (ROS), resulting in the formation of the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF), which serves as an indicator of oxidative stress within the cells. 2 ml microalgae sample isolated from the control, NaCl and NaCl+Na 2 SO 4 treated cultures were centrifuged at 12000 g for 5min at room temperature. Pellet were then washed with 1ml phosphate buffer saline (50 mM pH 7.4) twice. After incubating the samples in the dark for 1 hour with 20 µM H₂DCFDA dye (20 µL added per ml of PBS), fluorescence intensity was measured using a microplate reader spectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, using black 96-well microplates to minimize background interference Reactive oxygen species (ROS) levels were expressed in terms of relative fluorescence units (RFU). 5.7.2. Determination of Cell apoptosis Apoptosis was detected using the Annexin V- FITC staining kit (eBioscience™ Annexin V Apoptosis Detection Kit) [35]. 1ml microalgae sample isolated from the control, NaCl treated and NaCl + Na 2 SO 4 treated cultures were centrifuged at 12000 g for 5 min at room temperature, pellets are washed with 50mM phosphate buffer saline (pH 7.4). Annexin binding buffer and Annexin V- FITC were added to pellet and kept for the 20 minutes. The treated cells were centrifuged and again resuspend with annexin binding buffer and kept at 4°c. Cells were assessed after being kept in the dark for 1 hour and flow cytometer (BD Accuri™ C6 Plus) was used for analysis. 5.8. Statistical Analysis For each treatment, there were three replicates. The gathered data was statistically analysed using one-way ANOVA and Duncan's multiple range test to determine the significance of differences at p ≤ 0.05 between the treatments through IBM SPSS (Version 25). Declarations Ethical Approval Not applicable. Consent to Participate Not applicable. Consent for Publication All authors have given their consent to publish their work. Funding No funding available. Conflict of interest The authors declare that they have no conflict of interest. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors Contributions A.Y.: conceptualization, writing—original draft, writing draft—review and editing. L.A.S.: writing draft—review and editing. S.S.: data curation, writing draft—review and editing. R.B. and P.K.: writing draft—review and editing. P.S.: investigation, conceptualization, resources, data curation, writing—original draft, writing draft—review and editing, project administration. M.M.: investigation, conceptualization, resources, data curation, writing—original draft, writing draft—review and editing, project administration. All the authors reviewed and edited the contents. All authors have read and agreed to the published version of the manuscript. References Zhila, N. O., Kalacheva, G. S. & Volova, T. G. Effect of salinity on the biochemical composition of the alga Botryococcus braunii Kütz IPPAS H-252. J. Appl. Phycol. 23 , 47–52. https://doi.org/10.1007/s10811-010-9532-8 (2011). Khan, M. I., Shin, J. H. & Kim, J. D. The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb. Cell. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7804893","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":593671907,"identity":"03aa06a4-422c-44f0-b2f3-b77dfa9af6d1","order_by":0,"name":"Ankush Yadav","email":"","orcid":"","institution":"Central University of Punjab","correspondingAuthor":false,"prefix":"","firstName":"Ankush","middleName":"","lastName":"Yadav","suffix":""},{"id":593671908,"identity":"35f96ee9-2aa9-471e-be85-86e75260c793","order_by":1,"name":"Laishram Amarjit Singh","email":"","orcid":"","institution":"Central University of Punjab","correspondingAuthor":false,"prefix":"","firstName":"Laishram","middleName":"Amarjit","lastName":"Singh","suffix":""},{"id":593671909,"identity":"87ecee26-9c6a-4d9f-96b6-6d12e4248dbc","order_by":2,"name":"Suhani Sharma","email":"","orcid":"","institution":"Central University of Punjab","correspondingAuthor":false,"prefix":"","firstName":"Suhani","middleName":"","lastName":"Sharma","suffix":""},{"id":593671910,"identity":"fd07e318-5fbd-42ec-8649-0684be30ec7c","order_by":3,"name":"Rupesh Bhardwaj","email":"","orcid":"","institution":"Central University of Punjab","correspondingAuthor":false,"prefix":"","firstName":"Rupesh","middleName":"","lastName":"Bhardwaj","suffix":""},{"id":593671911,"identity":"6aa76e3f-1a86-4323-ba02-d9856304236a","order_by":4,"name":"Pritee Kumari","email":"","orcid":"","institution":"Central University of Punjab","correspondingAuthor":false,"prefix":"","firstName":"Pritee","middleName":"","lastName":"Kumari","suffix":""},{"id":593671912,"identity":"86ae5b31-f092-4877-ad33-3f212b673880","order_by":5,"name":"Prashant Swapnil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIie3RMUsDMRTA8ScHd8s7s6ZE/QyRg5zlvswVoV0qtos6lHJTXKSu7bcQCsWxEDiXuAs6FARnIdBFKObE1hbTgptD/tMj5JcEAuDz/dOCtZkekNVi4Ny9InRJsFb8kQDy6a+rN0uZejWd3ksfiD423fsTTB60mMFV1iiieOoi9UFTsGH5RoG2EzbSFIVupxx0q1EE+7mLcA0iwFBZEt+xWFryhILuSWUJcjeJjMGFJeRx/FGRZFiRxS6C3B5uCZxNvm7htCLFdlK/wQsWD1RN0vN5NrKE6uYlz8tWIreQFKOxwbkihJSnz13ZPyLXajJ772WHt0S7H7YcQvj5ixDy75WdBDaIz+fz+db6BM1HT5bpjMudAAAAAElFTkSuQmCC","orcid":"","institution":"Central University of Punjab","correspondingAuthor":true,"prefix":"","firstName":"Prashant","middleName":"","lastName":"Swapnil","suffix":""},{"id":593671913,"identity":"3bc35e78-47b4-4b9d-acb8-1912e6de418c","order_by":6,"name":"Mukesh Meena","email":"","orcid":"","institution":"Mohanlal Sukhadia University","correspondingAuthor":false,"prefix":"","firstName":"Mukesh","middleName":"","lastName":"Meena","suffix":""}],"badges":[],"createdAt":"2025-10-08 06:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7804893/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7804893/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103507050,"identity":"4d9f5f19-e0e7-4c5e-baeb-33bd245b305b","added_by":"auto","created_at":"2026-02-26 13:40:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66885,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth estimation of \u003cem\u003eChlorella sorokiniana\u003c/em\u003e (PV648655) under equimolar salt stress (350 mM NaCl) and mixed salt treatment (350 mM NaCl + Na₂SO₄). Data represent means ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/8bc4f66f8cf880e3a5ba3d2e.jpg"},{"id":103345511,"identity":"ddd64125-26d1-46d7-9c78-bb32ec0d34c2","added_by":"auto","created_at":"2026-02-24 16:12:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A).\u003c/strong\u003eChlorophyll yield under the same salt treatments. Different letters indicate statistically significant differences at P\u0026lt; 0.05, determined by Duncan’s multiple range test. Data are presented as means ± SD (n = 3).\u003cbr\u003e\n(B) Chlorophyll content (µg/mg dry cell weight) under equimolar NaCl and mixed salt stress. Different letters indicate significant differences at the 5% level (P \u0026lt; 0.05) using Duncan’s multiple range test. Values are means ± SD from three independent replicates.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/ffa4063c64977aab16f03097.jpg"},{"id":103345512,"identity":"cbc26f4b-4e87-40c8-a9d5-a0d1ec7d5f15","added_by":"auto","created_at":"2026-02-24 16:12:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect on the sucrose production under equimolar salt-stress 350 mM NaCl and salt mixture 350 mM (NaCl + Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sorokiniana \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003ePV648655)\u003cstrong\u003e. Values with different letters are significantly different at 5% level by Duncan's multiple range test.\u0026nbsp; Data are average ± SDs of three independent experiments (P\u0026lt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/624870cb1b610381f8c03a73.jpg"},{"id":103345514,"identity":"f071a0ad-03a6-4a30-8b8f-8256b4c543c8","added_by":"auto","created_at":"2026-02-24 16:12:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph illustrates the antioxidant enzyme activity (DPPH and SOD) under equimolar concentration of 350 mM NaCl and 350 mM (NaCl + Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. sorokiniana \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003ePV648655)\u003cstrong\u003e. Values with different letters are significantly different at 5% level by Duncan's multiple range test.\u0026nbsp; Data are average ± SDs of three independent experiments (P \u0026lt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/08b81928350ab0b348fa7db7.jpg"},{"id":103506707,"identity":"1f7092da-306d-4161-b108-91306c3188cd","added_by":"auto","created_at":"2026-02-26 13:39:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37394,"visible":true,"origin":"","legend":"\u003cp\u003eImage of Native gel representing the isozymes bands of SOD under different salt concentrations. Arrow indicating the 4\u003csup\u003eth\u003c/sup\u003e additional band. (A) Control, (B) 350 mM NaCl, (C) 350 mM (\u003cstrong\u003eNaCl + Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e). The arrow represents the additional fourth band under 350 mM NaCl.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/649c369725493bd4120c981a.jpg"},{"id":103345518,"identity":"cee7ce83-2f02-4091-8f5a-fa2e7e2407f0","added_by":"auto","created_at":"2026-02-24 16:12:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":88833,"visible":true,"origin":"","legend":"\u003cp\u003eImage of SDS gel showing protein bands under different salt concentrations. Yellow arrow – Downregulation of protein bands. Red arrow – Upregulation of protein bands.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/8e824cbfadbef1958dac0798.jpg"},{"id":103345515,"identity":"81954141-882d-44bd-8d93-13c9ebcfc1fd","added_by":"auto","created_at":"2026-02-24 16:12:35","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":73726,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of \u003cem\u003eC. sorokiniana \u003c/em\u003e(PV648655)\u003cem\u003e \u003c/em\u003ealgal cells intracellular lipid accumulation using confocal microscopy under different salt stress in which (A) Control, (B) 350 mM NaCl, (C) 350 mM (NaCl + Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). Image captured at 530 nm excitation and 575 nm emission wavelengths. The green regions represent chlorophyll fluorescence, while the red-colored cells exhibit autofluorescence.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/41de4a622af653a478ab4522.jpg"},{"id":103345520,"identity":"35a3f2a5-5a45-4ef8-a266-f04f522dd69f","added_by":"auto","created_at":"2026-02-24 16:12:35","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":56985,"visible":true,"origin":"","legend":"\u003cp\u003eGraph illustrate the ROS levels under\u003cstrong\u003e \u003c/strong\u003e350mM NaCl \u0026amp; 350mM NaCl+Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e salt stress. \u003cstrong\u003eValues with different letters are significantly different at 5% level by Duncan's multiple range test.\u0026nbsp; Data are average ± SDs of three independent experiments (P \u0026lt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/fe8f528a01e1b41642721b40.jpg"},{"id":103345517,"identity":"5ca496e4-0b01-40ee-b03b-f6fb93203d0a","added_by":"auto","created_at":"2026-02-24 16:12:35","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":108525,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric detection of cell death by Annexin V method in microalgae cells where (a) Control (b) 350mM NaCl stress (c) 350mM NaCl + Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/541aa97af7a487fa248722fc.jpg"},{"id":104397516,"identity":"32241322-a9c6-4182-8ae8-142cf67e3a44","added_by":"auto","created_at":"2026-03-11 11:50:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2216649,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/96587d6e-f741-4b32-9466-ce2f428aad40.pdf"},{"id":103506996,"identity":"f23959a0-4f38-4c22-87ba-585595d1c6df","added_by":"auto","created_at":"2026-02-26 13:40:10","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":332800,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.doc","url":"https://assets-eu.researchsquare.com/files/rs-7804893/v1/818e2915a5fd3fdd9a696821.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physiological resilience of freshwater microalgae Chlorella sorokiniana under NaCl stress supplemented with sodium sulfate as a mitigating agent","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicrobes play an essential biogeochemical role in the ecosystem. Microalgae are attracting study interest because of their capacity to produce diverse substances, rapidly increase biomass, and alter their biochemical composition in response to changing growth circumstances [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Microalgae, due to their ability to efficiently use carbon dioxide and water, make them sustainable in the soil where traditional crops are unsuitable [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Salinity has a substantial effect on the development and productivity of microbial communities such as microalgae, fungi, and bacteria. It changes ionic balance and osmotic potential, affecting photosynthesis, respiration, and nutrient intake [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Microalgae, having a remarkable tolerance to different abiotic stresses, employ various physiological and biochemical strategies to thrive and prosper in a saline environment [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Microalgae in stress conditions have been found to produce more lipid content. Some microalgae, such as \u003cem\u003eDesmodesmus abundans and Chlorella pyrenoidosa\u003c/em\u003e, have been found to produce higher lipid content when exposed to certain concentrations of salt stress [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Different concentrations of salt affect growth, biomass yield and lipid accumulation of \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eScenedesmus obliquus\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Sodium chloride (NaCl) and Sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) are often used to adjust salinity levels. NaCl being cost-effective and user-friendly, is often used to induce lipid content in microalgae [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the high lipid content produced by this procedure affects the biomass quantity and thus overall lipid production. Therefore, it is necessary to overcome this contradiction and develop cultures that allow optimal growth while also producing high lipid content.\u003c/p\u003e \u003cp\u003eSupplementing with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e has been reported to increase the production of biomass and promote growth in species such as \u003cem\u003eChlamydomonas moewusii\u003c/em\u003e and \u003cem\u003eSpirulina platensis\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], \u003cem\u003eScenedesmus quadricauda\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The synergistic effects of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e on the development and productivity of microalgae remain little understood. This research aims to examine the impact of individual salt (NaCl) and combination salt (NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) on protein expression, osmolyte levels, and antioxidant concentration levels in microalgae.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Isolation and Characterization of \u003cem\u003eChlorella sorokiniana\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMorphological observation under a light microscope revealed characteristic spherical to oval-shaped cells typical of \u003cem\u003eChlorella\u003c/em\u003e species (Supplementary Fig.\u0026nbsp;1). The result of PCR showed an amplified gene fragment length of approximately 1800 bp (Supplementary Fig.\u0026nbsp;2). The resulting sequence was subjected to BLAST analysis using the NCBI database, which confirmed the strain as \u003cem\u003eChlorella sorokiniana\u003c/em\u003e, which is highly homologous to \u003cem\u003eChlorella sorokiniana\u003c/em\u003e isolate NS5 (KM502980.1) with 99.09% sequence similarity. The sequence was subsequently submitted to the NCBI GenBank and received accession number PV648655. The phylogenetic tree was constructed and confirmed the organism as \u003cem\u003eChlorella sorokiniana\u003c/em\u003e (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2. Estimation of Growth and Chlorophyll Content\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe microalgae were grown in BG-11 medium with equimolar concentrations of 350 mM (NaCl) and 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), along with a control condition. Growth analysis showed better growth in the culture treated with 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) as compared to the culture treated with 350 mM (NaCl), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The specific growth rate (\u0026micro;), division rate per day (Dd), and doubling time (tD) all followed a consistent trend, showing a 2.74-fold decline in \u0026micro; and Dd and a 2.74-fold increase in tD under 350 mM NaCl treatment, while the combined treatment with 350 mM NaCl\u0026thinsp;+\u0026thinsp;Na₂SO₄ exhibited a lesser impact, with a 1.75-fold reduction in \u0026micro; and Dd and a 1.75-fold increase in tD compared to the control. In terms of biomass productivity, a significant 4.86-fold decrease was observed under NaCl stress, whereas the combined salt treatment resulted in a comparatively lower 2.54-fold reduction, indicating partial mitigation of salt-induced growth inhibition by sodium sulfate supplementation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Chlorophyll yield was estimated on the 8th day, which showed the high chlorophyll yield in the control, followed by salt mixture 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), and last 350 mM (NaCl) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (A). Chlorophyll yield per mg of dry cell weight was also estimated. The culture treated with 350 mM NaCl yielded approximately only 27% compared to the control, while the culture exposed to combined salt treatment retained approximately 56% of the chlorophyll yield relative to the control, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (B). While both salts induced stresses leading to a decrease in chlorophyll yield, the combination of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e showed better chlorophyll than that of the culture with only NaCl. The presence of SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026oline;\u003c/sup\u003e may possibly lessen the salt toxicity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth Kinetics of Chlorella sorokiniana Under 350 mM NaCl and 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na₂SO₄) Treatments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecific Growth Rate (\u0026micro;) (day⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDivision per Day (Dd) (div\u0026middot;day⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDoubling Time (tD) (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBiomass Productivity (cells\u0026middot;mL⁻\u0026sup1;\u0026middot;day⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c6\"\u003e \u003cp\u003e4.119 \u0026times; 10⁶\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e350 mM NaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c6\"\u003e \u003cp\u003e8.473 \u0026times; 10⁵\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na₂SO₄)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1732\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c6\"\u003e \u003cp\u003e1.6219 \u0026times; 10⁶\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sucrose Estimation\u003c/h2\u003e \u003cp\u003eSucrose content was quantified to evaluate osmolyte accumulation in \u003cem\u003eChlorella sorokiniana\u003c/em\u003e (PV648655) cultures exposed to varying concentrations of salt stress. Measurements were conducted on alternate days using a sucrose standard curve for calibration. The level of cellular sucrose production significantly increased with an increase in salt exposure time and attained its maximum on the 8th and 10th days of salt exposure. The increment in sucrose production was 2.9 and 2.29 folds noted in cells exposed to 350 mM NaCl and 1.6 and 1.61 folds in cells exposed to a salt mixture of 350 mM (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NaCl) in comparison to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antioxidant Enzyme Activity and DPPH Radical Scavenging Assay\u003c/h2\u003e \u003cp\u003eThe antioxidant activity in \u003cem\u003eC. sorokiniana\u003c/em\u003e (PV648655) cells exposed to different concentrations of salt stress was assessed using the DPPH radical scavenging assay and superoxide dismutase (SOD) activity measurement. After 72 hours of salt stress, DPPH radical scavenging activity and SOD activity were found to be 1.71- and 1.95-fold higher, respectively, in cells treated with 350 mM NaCl compared to the control. In cells treated with a 350 mM salt mixture (NaCl\u0026thinsp;+\u0026thinsp;Na₂SO₄), these values increased by 1.38- and 1.10-fold, respectively, compared to the control as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Native PAGE of Superoxide Dismutase\u003c/h2\u003e \u003cp\u003eIsozyme profiling of antioxidative enzymes showed three isoforms (61.9, 56.5, and 51.9 kDa) of SOD in control and salt-stressed cells. The intensity of bands was high in cells with 350 mM NaCl and a salt mixture of 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). An additional band of isozyme was found in cells treated with NaCl of molecular weight between 61.9 and 60 kDa, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Isozymes are enzymes that catalyze the same reaction but have a different molecular formula.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. SDS PAGE\u003c/h2\u003e \u003cp\u003eThe result of SDS PAGE showed that the protein bands upregulation was higher in equimolar concentration 350 mM NaCl-treated cells than in 1:1 mixture-treated cells with respect to the control. Downregulation of protein bands was high in the cells treated with a 1:1 salt mixture with respect to the control. The bands of molecular weight 168.5, 141.7, 101.8, 69.9, and 53.3 kDa were highly downregulated in the 1:1 salt mixture, respectively, to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Nile Red Assay\u003c/h2\u003e \u003cp\u003eIntracellular lipid accumulation was observed using Nile red dye. Accumulation of lipids was higher in the cells treated with 350 mM NaCl than in cells treated with the salt mixture (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Recent studies discussed under stress condition increament of ROS are closely related to total lipid content in microalgae but the exact mechanism behind this phenomenon remains to be explore [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The green images are due to chlorophyll and red colour cells showing autofluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. ROS detection\u003c/h2\u003e \u003cp\u003eReactive oxygen species (ROS) levels, indicating total cellular oxidative stress, were assessed using the H₂DCFDA dye, and the relative fluorescence intensity was recorded, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Under 350 mM NaCl stress, fluorescence intensity increased by 1.63-fold compared to the control, while cells exposed to 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na₂SO₄) showed a 1.29-fold increase. Notably, ROS levels in the combined salt treatment were reduced by 1.26-fold relative to NaCl alone. These findings indicate that NaCl stress induces excessive ROS accumulation, leading to potential cytotoxicity. In contrast, the reduced ROS generation under the combined salt stress suggests a protective effect of sulfur, partially mitigating oxidative stress. This supports the role of sulfur supplementation in alleviating the harmful effects of salinity-induced oxidative damage in microalgae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Cell Apoptosis detection by Annexin-V FITC\u003c/h2\u003e \u003cp\u003eFlow cytometry analysis showed that under control conditions, 96.3% of the cells (LL region) were viable, with 2.8% in the early apoptotic stage (LR region) and 0.4% in the late apoptotic stage (UR region). Under 350 mM NaCl stress, cell viability decreased to 91.6%, while early and late apoptotic cells increased to 6.7% and 1.0%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In contrast, cells exposed to the combined salt stress (350 mM NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO₄) showed slightly improved viability at 93.4%, with 4.9% early apoptotic and 0.8% late apoptotic cells. These results suggest that sulfur supplementation reduces salt-induced apoptosis and helps maintain higher cell viability.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eSalinity suppresses the growth and yield of \u003cem\u003eC. sorokiniana\u003c/em\u003e (PV648655); however, NaCl alone induced more toxicity in comparison to the equimolar concentration of the NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e mixture. In a similar experiment conducted in \u003cem\u003eAnabaena fertilissima\u003c/em\u003e, it was found that oxidative stress is lower in those cells exposed to the salt mixture in comparison to those cells exposed to NaCl only [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In stressed conditions, antioxidant molecules such as glutathione, thioredoxin, and glutaredoxin are rapidly synthesized to mitigate stress and facilitate the normal development of microalgae [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The presence of SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the mixture might reduce the stress as SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e can be readily incorporated into protein as the redox-active cysteine residue and be used in synthesizing the antioxidants [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It has been found that sulfate in mustard increases glutathione production, which in turn increases salt tolerance [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] while its deprivation has been found to reduce glutathione content [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChlorophyll yield can be used as an indicator of photosynthetic health in \u003cem\u003eC. sorokiniana\u003c/em\u003e PV648655. High salinity leads to ionic and osmotic imbalances, disrupting photosystem and chlorophyll synthesis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The negative effect of salt on microalgal proliferation was further confirmed by the growth kinetics data, indicating partial mitigation of salt toxicity. This highlights the role of sulfate in maintaining better growth dynamics even under high saline conditions, possibly by supporting key metabolic and stress-alleviation pathways. The chlorophyll yield under the combined salt condition (350 mM NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) was found to be higher than in the 350 mM NaCl treatment alone. It may be because SO\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e\u0026ndash;\u003c/sup\u003e might have mitigated some of the detrimental effects caused by high Na\u003csup\u003e+\u003c/sup\u003e. The presence of SO\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e\u0026ndash;\u003c/sup\u003e likely helps in maintaining cellular integrity under osmotic stress by contributing to the synthesis of osmoprotective molecules [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe algae employ a ROS-scavenging strategy to fight higher production of ROS, which includes enzymatic and non-enzymatic mechanisms [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Salinity increases the activity of antioxidants such as SOD, CAT, and GR to overcome oxidative stress and maintain cellular redox homeostasis at its optimum [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The antioxidative enzyme activity significantly increased in the cells exposed to 350 mM NaCl compared to 350 mM NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e exposed cells, suggesting \u003cem\u003eC. sorokiniana\u003c/em\u003e experiences elevated levels of ROS under high salinity. The lower antioxidative enzyme activity in the equimolar mixture of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e exposed cells suggests that the presence of SO\u003csub\u003e4\u003c/sub\u003eˉ could help in reducing oxidative stress. It has been reported that sulphur is the precursor of the synthesis of sulfur-containing antioxidants such as glutathione and other thiol compounds, which are major cellular antioxidants and detoxify ROS to maintain the redox balance. Therefore, the presence of sulfate enhances the cell\u0026rsquo;s ability to cope with oxidative stress [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The uptake of ions by microalgal cells follows the specific membrane transport systems, located in the plasma membrane. Na⁺ enters the cell via Na⁺/H⁺ antiporters, which help to regulate ion homeostasis and osmotic balance under saline or stress conditions [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In case of Cl⁻, transport through anion channels and H⁺/Cl⁻ symporters, to maintain turgor pressure [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. While SO₄\u0026sup2;⁻ are taken up through the sulfate transporter. This helps to mediate sulfate uptake for assimilation into sulfur-containing amino acids like cysteine and methionine [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. (Nagesh et al., 2024). The result of the experiment points to a potential protective mechanism where SO\u003csub\u003e4\u003c/sub\u003eˉ supports cellular redox balance, thereby lessening the dependence on enzymatic antioxidants.\u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD) isozyme profiling under conditions of salt stress demonstrates a multifaceted and adaptable antioxidative response in cells. An elevated oxidative stress response to NaCl is shown by the detection of three SOD isoforms (61.9 kDa, 56.5 kDa, and 51.9 kDa) in both salt-stressed and control cells, with greater band intensity in cells treated with 350 mM NaCl. The discovery of a second SOD isoform that is only present in cells treated with NaCl and ranges in size from 61.9 to 60 kDa points to the induction of a novel isozyme that is only responsive to stress caused by NaCl. These results highlight the functions of various SOD isozymes in oxidative stress management, including Cu/Zn-SOD in the cytosol and chloroplasts, Mn-SOD in mitochondria and Fe-SOD in the cytoplasm and chloroplasts [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe observed increase in total soluble protein content and altered banding pattern in SDS-PAGE under NaCl\u0026thinsp;+\u0026thinsp;Na₂SO₄ treatment suggests that the combined salt stress altered intracellular protein expression patterns, possibly through modulation of stress signaling and sulfur assimilation pathways. The presence of SO₄\u0026sup2;⁻ may support enhanced synthesis of stress-related proteins by improving redox balance, sulfur-containing amino acid availability (e.g., cysteine, methionine), and antioxidant defense, rather than directly affecting protein solubility in the medium [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Therefore, the differences in protein band intensity are attributed to changes in cellular stress physiology, not to extracellular ionic effects on solubility. The addition of NaCl increases the ionic strength of the solution, leading to the \"salting out\" effect, where proteins become less soluble and may precipitate out of the solution [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This results in a lower observed protein concentration (1.576 \u0026micro;g/\u0026micro;l). When Na₂SO₄ is added along with NaCl, the solution contains both Na⁺, Cl⁻, and SO₄\u0026sup2;⁻ ions. Sulfate ions affect enhancing protein solubility by stabilizing the hydration shell around proteins and reducing protein-protein interactions that lead to aggregation and precipitation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This stabilization effect counteracts the salting-out effect of NaCl, leading to a higher observed protein concentration (3.882 \u0026micro;g/\u0026micro;l). The observed increase in protein concentration is due to electrostatic stabilization, hydration shell formation, and charge screening. NaCl alone promotes \"salting out\" and reduces protein solubility, while the addition of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e introduces sulfate ions that enhance protein solubility through various stabilizing interactions, leading to a net increase in the observed protein concentration in the NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution.\u003c/p\u003e \u003cp\u003eResults observed through the Nile red assay showed that lipid accumulation was notably higher in cells treated with 350 mM NaCl compared to those in the combined salt treatment. The difference in intracellular lipid accumulation between cells treated with 350 mM NaCl alone and those treated with 350 mM NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e likely arises from the varying effects of these salts on cellular processes and metabolism. High concentrations of NaCl can induce osmotic stress, triggering various cellular responses, including changes in gene expression and metabolism. This stress can activate signaling pathways such as the MAPK and NF-κB pathways, which regulate cellular responses to stress, including inflammation and lipid metabolism. Additionally, osmotic stress can lead to increased lipogenesis, resulting in enhanced lipid accumulation within cells [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. High salt concentrations can disrupt cellular homeostasis, including ion balance and energy metabolism, further contributing to changes in lipid metabolism and accumulation. Lipid bodies can sequester excess ROS and act as energy reservoirs during stress conditions. The high lipid accumulation in NaCl alone-treated cells suggests that microalgae channel resources toward lipid synthesis to cope with oxidative damage. The addition of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e introduces sulfate ions into the cellular environment, potentially altering cellular processes. The presence of sulfate ions alongside NaCl may moderate osmotic stress, reducing the extent of cellular responses associated with osmotic stress, including changes in lipid metabolism. The reduced apoptosis observed under combined salt treatment likely reflects the role of sulfur in reinforcing cellular defense and maintaining membrane integrity under stress conditions. Once sulfate ions are taken up by specific transporters they are assimilated into cysteine through a tightly regulated sulfur assimilation pathway. Cysteine serves as a precursor for glutathione and other thiol-containing antioxidants, which are pivotal in detoxifying ROS and stabilizing redox homeostasis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. By supporting the biosynthesis of such protective molecules, sulfur reduces cellular damage, prevents oxidative-triggered programmed cell death, and enhances overall stress resilience. This protective mechanism parallels the concept of sulfur-enhanced defense (SED) observed in higher plants [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], suggesting that in \u003cem\u003eChlorella sorokiniana\u003c/em\u003e, sulfur availability not only contributes to metabolic demands but also actively modulates cellular survival pathways under abiotic stress.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e \u003cem\u003eChlorella sorokiniana\u003c/em\u003e exhibited alterations in protein expression under salt stress conditions. However, the combined treatment of NaCl and Na₂SO₄ alleviated the adverse effects induced by NaCl alone. The presence of Na₂SO₄ appeared to mitigate NaCl-induced stress, as evidenced by a reduction in antioxidant activity compared to cultures treated solely with NaCl. The findings conclude that Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e is a potent mitigating agent for salt (NaCl) stress in \u003cem\u003eC. sorokiniana\u003c/em\u003e, this underscore the adaptive mechanisms of \u003cem\u003eC. sorokiniana\u003c/em\u003e. Further, research should aim to clarify the molecular signalling pathways involved in the salt stress responses and explore the potential of combining different salts to boost the resilience and productivity of microalgae.\u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e5.1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eIsolation and Characterization of \u003cem\u003eChlorella sorokiniana\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh water microalgal samples collected from the pond of Guru Nanak Dev Thermal Power Plant, Bhatinda, India. Samples were transferred to BG-11 media to obtain an axenic strain as described in the methods [65]. The isolated strain was observed under a light microscope (Nikon Eclipse Ci-L plus) using a 60× objective lens with bright-field contrast to examine its morphological features. Genomic DNA was isolated using the CTAB method [66] for molecular characterization and amplified by polymerase chain reaction (PCR) using primers targeting the 18S ribosomal RNA gene region. The primer sequences used were: forward (18S-F) 5′-GTCATATGCTTGTCTCAAAGATTAAGCC-3′ and reverse (18S-R) 5′-CCTTGTTACGACTTCTCCTTCCTCTAA-3′. The amplified product was analyzed using the NCBI BLAST (Basic Local Alignment Search Tool) for taxonomic identification and subsequently submitted to the NCBI database to obtain an accession number.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2. Growth Measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.1. Growth Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were carried out in the 500 ml conical flask containing 300 ml BG-11 culture media to study the impact of NaCl, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e + NaCl salt [67]. First, the exponential phase culture (300 ml) of \u003cem\u003eC. sorokiniana\u0026nbsp;\u003c/em\u003ewas centrifuged for 5 minutes at 7000 g at 25\u003csup\u003eo\u003c/sup\u003eC and subsequently introduced into flasks containing equimolar concentrations of 350 mM NaCl, 350 mM (NaCl + Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), along with the culture in the control condition. Further, the cultures were maintained in a culture chamber set at 28 ± 1\u003csup\u003eo\u003c/sup\u003eC and under a 16:8 h light:dark photoperiod with a light intensity of 50 µmol/m²/s throughout the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.2. Estimation of Growth and Chlorophyll Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.2.1. Optical Density-Based Growth Monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth of \u003cem\u003eChlorella sorokiniana\u003c/em\u003e was monitored daily by measuring optical density at 750 nm using a double-beam UV-Visible spectrophotometer (Motras Scientific, India). The culture reached an OD₇₅₀ of 1.0 on the 8th day of cultivation, which was used as the reference point for further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.2.2. Growth Kinetics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA standard curve of optical density (OD₇₅₀) versus cell count was generated using \u003cem\u003eC. sorokiniana\u003c/em\u003e cultures at varying cell densities. Cell enumeration was performed using a hemocytometer (Improved Neubauer chamber) following the protocol described by Zhang (2020) [68]. To ensure data reliability, all cell counts were conducted in triplicate.\u003c/p\u003e\n\u003cp\u003eGrowth kinetics were evaluated by calculating the specific growth rate (μ), division rate per day (Dd), doubling time (td), and biomass productivity (P), as described by Gani et al., (2016) [69], using the following equations:\u003c/p\u003e\n\u003cp\u003eSpecific growth rate (µ/day) = ln (Nf/Ni)/ Tf-Ti\u003c/p\u003e\n\u003cp\u003eDivision per day (Dd) = µ/day / ln2\u003c/p\u003e\n\u003cp\u003eDoubling time (td) = 1/ Dd\u003c/p\u003e\n\u003cp\u003eBiomass productivity (cell/mL/day) = (Nf/Ni)/ Tf-Ti\u003c/p\u003e\n\u003cp\u003eWhere Ni and Nf are the initial and final cell concentrations (cells·mL⁻¹), and Ti and Tf are the respective time points (in days). The exponential growth phase was estimated using a minimum of three time points, plotting the growth curves for parameter estimation accurately.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.2.3. Chlorophyll Content Estimation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll (chl) estimation was carried out at this point, following the procedure described by Kirk \u0026amp; Allen (1965) [70]. For the analysis, 5 ml of microalgal culture was centrifuged at 9000 × g for 10 minutes in a 15 ml centrifuge tube. The supernatant was discarded, and the pellet was resuspended in 5 ml of 80% acetone, thoroughly mixed by vortexing, and incubated overnight in the dark at 4°C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter centrifugation, chl µg/ml was calculated using a spectrophotometer to measure the absorbance at 663.6 nm and 646.6 nm against acetone as blank. The concentration of Chlorophyll a and Chlorophyll b were evaluated according to the following equations [71].\u003c/p\u003e\n\u003cp\u003eChlorophyll a (µg chlorophyll/ml medium) = (12.25E\u003csub\u003e663.6\u0026nbsp;\u003c/sub\u003e– 2.55E\u003csub\u003e646.6\u003c/sub\u003e) / V\u003c/p\u003e\n\u003cp\u003eChlorophyll b (µg chlorophyll/ml medium)\u0026nbsp;= (20.31E\u003csub\u003e646.6\u0026nbsp;\u003c/sub\u003e– 4.91E\u003csub\u003e663.6\u003c/sub\u003e) / V\u003c/p\u003e\n\u003cp\u003eChlorophyll a+b (µg chlorophyll/ml medium)\u0026nbsp;= (17.76E\u003csub\u003e646.6\u0026nbsp;\u003c/sub\u003e+ 7.34E\u003csub\u003e663.6\u003c/sub\u003e) / V\u003c/p\u003e\n\u003cp\u003eWhere:\u003c/p\u003e\n\u003cp\u003eE\u003csub\u003e663.6\u0026nbsp;\u003c/sub\u003eand E\u003csub\u003e646.6\u0026nbsp;\u003c/sub\u003erepresent absorbances at 663.6 nm and 646.6 nm minus absorbance at 750 nm, respectively\u003c/p\u003e\n\u003cp\u003eV = Volume of the sample (ml)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3. Estimation of sucrose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEstimation of sucrose was done using the method Van Handel (1968) [72]. 50 ml of microalgal culture were centrifuged at 7000 g at 25°C for 5 min. Pellets were homogenized in 80% ethanol to extract soluble sugars and kept in a water bath set at 100°C for 5–10 minutes. 100 µL of 30% KOH were added to the residue and again kept in a water bath at 100°C for 5 minutes. The samples were allowed to cool to room temperature, and then 3 ml of anthrone reagent was added and warmed up at 40°C for 15 minutes. The samples were allowed to cool down at room temperature, absorbency was recorded at 620 nm wavelength. \u0026nbsp;Sucrose concentrations were calculated by comparing the absorbance values to a standard curve prepared with known concentrations of sucrose processed identically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4. Determination of Antioxidative Enzyme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.1. DPPH Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntioxidant activity was assayed by the method of Dawidowicz et al., (2012) [73], using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The standard was prepared for the DPPH assay against ascorbic acid of a known different concentration. A stock solution of 1 mM DPPH (methanol) and an ascorbic concentration (5–70 μg/ml) was prepared in methanol. The absorbance was recorded at 517 nm after 30 minutes of incubation in the dark on a microplate reader (Synergy H1, BIOTEK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.2. Quantification of Superoxide Dismutase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide dismutase (SOD) activity was determined by measuring the inhibition of photochemical reduction of the nitro blue tetrazolium (NBT) method by Beauchamp \u0026amp; Fridovich (1971) [74]. In a test tube, a 1 ml reaction mixture containing phosphate buffer (50 mM pH -7.8), 9.9 mM methionine, 0.025% Triton-X 100, 57 μ mol/l NBT, and 20 μl extracted enzyme was added. After that, 10 μl of riboflavin (4.4 mg/100 ml) was added. The tubes were kept under 20-watt fluorescent bulbs. After 15 minutes, the absorbance was recorded at 560 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.3. Native PAGE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to ascertain the expression of distinct antioxidant enzyme isozymes, an equal amount of protein extract was loaded onto 10% native polyacrylamide gel and electrophoresed at a constant voltage of 150 V at 4\u003csup\u003eo\u003c/sup\u003eC till the dye reached the bottom of the gel method as described by Sambrook and Russell (2001) [75]. The image of the gel was captured by gel-doc after enzyme-specific staining of the gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.5. Protein Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein was extracted in accordance with Hurkman and Tanaka (1986) [76]. The exponential growth of microalgae culture was centrifuged at 9000 × g for 15 min. The pellet was lyophilized and crushed into a powder using liquid nitrogen. Crushed cells were suspended in 2 ml of protein extraction buffer (0.7 M sucrose, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCL, 2 mM PMSF (phenylmethanesulfonyl fluoride), and 0.005% Triton-X). The mixture was collected in a microcentrifuge tube and centrifuged at 3040 g for 50 minutes at 4\u003csup\u003eo\u003c/sup\u003eC. The supernatant containing soluble protein was collected in a separate tube and stored at -20 \u003csup\u003eo\u003c/sup\u003eC for further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.5.1. SDS PAGE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn equal concentration (50μg/μl) of extracted protein was loaded onto SDS PAGE 10% (Sambrook and Russell, 2001) [75] along with a protein marker (10- 245 kDa), and the gel was run at 100 V for the stacking gel and 150 V for the resolving gel. The gel was removed when the dye reached its bottom and stained with Coomassie brilliant blue (CBB) R-250.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.6. Nile Red Assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNile red assay was done by the method of Zhao et al. (2019) [77]. 1 ml of culture was taken and centrifuged at 1137 × g for 3 minutes. The supernatant was discarded, and the pellet was resuspended in 200 μl of 20% DMSO and kept for 2 hours at room temperature. The suspension was again centrifuged at 1137 x g for 3 minutes, and a pellet was taken and resuspended in water. The prepared stock solution of Nile Red (1 mg/ml of absolute acetone). Nile red (5μl) was added to the algal cells and incubated for 10 minutes in the dark. The image of algal cells was taken at 530 nm excitation and 575 nm emission wavelengths.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.7. Determination of cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.7.1. ROS determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular ROS detection was done using 2’7’-dichlorodihydrofluorescein diacetate (H\u003csub\u003e2\u003c/sub\u003eDCFDA) dye [78]. The non-fluorescent dye H\u003csub\u003e2\u003c/sub\u003eDCFDA is oxidized by intracellular reactive oxygen species (ROS), resulting in the formation of the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF), which serves as an indicator of oxidative stress within the cells. 2 ml microalgae sample isolated from the control, NaCl and NaCl+Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treated cultures were centrifuged at 12000 g for 5min at room temperature. Pellet were then washed with 1ml phosphate buffer saline (50 mM pH 7.4) twice. After incubating the samples in the dark for 1 hour with 20 µM H₂DCFDA dye (20 µL added per ml of PBS), fluorescence intensity was measured using a microplate reader spectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, using black 96-well microplates to minimize background interference Reactive oxygen species (ROS) levels were expressed in terms of relative fluorescence units (RFU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.7.2. Determination of Cell apoptosis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptosis was detected using the Annexin V- FITC staining kit (eBioscience™ Annexin V Apoptosis Detection Kit) [35]. 1ml microalgae sample isolated from the control, NaCl treated and NaCl + Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treated cultures were centrifuged at 12000 g for 5 min at room temperature, pellets are washed with \u0026nbsp; 50mM phosphate buffer saline (pH 7.4). Annexin binding buffer and Annexin V- FITC were added to pellet and kept for the 20 minutes. The treated cells were centrifuged and again resuspend with annexin binding buffer and kept at 4°c. Cells were assessed after being kept in the dark for 1 hour and flow cytometer (BD Accuri™ C6 Plus) was used for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.8. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each treatment, there were three replicates. The gathered data was statistically analysed using one-way ANOVA and Duncan's multiple range test to determine the significance of differences at p ≤ 0.05 between the treatments through IBM SPSS (Version 25).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have given their consent to publish their work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.Y.: conceptualization, writing—original draft, writing draft—review and editing. L.A.S.: writing draft—review and editing. S.S.: data curation, writing draft—review and editing. R.B. and P.K.: writing draft—review and editing. P.S.: investigation, conceptualization, resources, data curation, writing—original draft, writing draft—review and editing, project administration. M.M.: investigation, conceptualization, resources, data curation, writing—original draft, writing draft—review and editing, project administration. All the authors reviewed and edited the contents. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhila, N. O., Kalacheva, G. S. \u0026amp; Volova, T. G. Effect of salinity on the biochemical composition of the alga \u003cem\u003eBotryococcus braunii\u003c/em\u003e K\u0026uuml;tz IPPAS H-252. \u003cem\u003eJ. Appl. 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Plant Sci.\u003c/em\u003e, \u003cb\u003e12\u003c/b\u003e, p.752634 .\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microalgae, Chlorella sorokiniana, NaCl, Na2SO4, Isozymes, Antioxidants","lastPublishedDoi":"10.21203/rs.3.rs-7804893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7804893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroalgae show remarkable resistance to abiotic stresses such as salt. This work investigates the effects of sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) and sodium chloride (NaCl) on the freshwater microalgal strain \u003cem\u003eChlorella sorokiniana\u003c/em\u003e with particular emphasis on physiological activities. The study is focused on to understand the molecular processes of \u003cem\u003eC. sorokiniana\u003c/em\u003e in a controlled environment using NaCl and equimolar concentrations of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Certain biochemical assays were resulted \u003cem\u003eC. sorokiniana\u003c/em\u003e, cultured with a salt mixture of 350 mM (NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), showed better growth than 350 mM NaCl. The ROS production was high in 350 mM NaCl, which is 1.71 and 1.95 times higher compared to the control and equimolar concentration of NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4,\u003c/sub\u003e respectively. The osmolyte level in 350 mM NaCl increased by 2.9 and 2.29-fold as compared to the control and equimolar concentration of NaCl\u0026thinsp;+\u0026thinsp;Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, respectively. Significant alterations in protein expression suggested that stress-response pathways have been activated. Furthermore, significant fluctuations in omolyte and antioxidant levels indicate the significant adaptation of microalga to salinity stress. These findings contribute to the optimization of microalgae farming by shedding light on the physiological and biochemical strategies of microalgae in saline environments and essential to comprehend in order to advance biotechnological applications that are sustainable.\u003c/p\u003e","manuscriptTitle":"Physiological resilience of freshwater microalgae Chlorella sorokiniana under NaCl stress supplemented with sodium sulfate as a mitigating agent","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 16:12:29","doi":"10.21203/rs.3.rs-7804893/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-19T09:56:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-14T12:59:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154713880703564528469438232213022067131","date":"2026-03-10T06:45:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T17:38:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T06:33:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243998540810825454807702849638000748325","date":"2026-02-19T05:17:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133977349765875151729909032988541066877","date":"2026-02-19T05:00:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87575643353920058683192436510917002369","date":"2026-02-17T04:43:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-17T04:37:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-13T12:53:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-09T09:44:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-09T09:41:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-08T06:50:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"88ff4304-c145-4afd-9ef7-cfbb834e9c4f","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63174273,"name":"Biological sciences/Biochemistry"},{"id":63174274,"name":"Biological sciences/Biotechnology"},{"id":63174275,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-12T07:54:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 16:12:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7804893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7804893","identity":"rs-7804893","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}