Oxidative stress enhances lipid production and improves fatty acid composition for biodiesel production in microalga Nannochloropsis oceanica

Oxidative stress enhances lipid production and improves fatty acid composition for biodiesel production in microalga Nannochloropsis oceanica | 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 Research Article Oxidative stress enhances lipid production and improves fatty acid composition for biodiesel production in microalga Nannochloropsis oceanica Majid Mahdieh, Abolfazl Taghavi, Mona Nezarat This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6458745/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The depletion of fossil fuel reserves, escalating energy prices, rising global energy demand, and heightened awareness of climate change have accelerated efforts to develop sustainable biofuel alternatives. Microalgae are considered a promising candidate for biodiesel production due to their rapid biomass accumulation, high lipid content, and superior photosynthetic efficiency compared to conventional terrestrial crops. This study examined the effect of oxidative stress, induced by hydrogen peroxide (H₂O₂), on the growth and lipid productivity of the marine microalga Nannochloropsis oceanica . Five concentrations of H₂O₂ (0, 100, 200, 300, and 400 µM) were applied during the exponential growth phase. Cultures were maintained under controlled conditions, and at the end of the cultivation period, biomass was harvested and freeze-dried. Lipids were extracted using a modified Bligh and Dyer protocol, and fatty acid profiles were determined through gas chromatography. The results demonstrated a significant enhancement in lipid accumulation under oxidative stress, with the highest lipid content—a 64% increase over the control—recorded at 400 µM H₂O₂. However, biomass productivity decreased at elevated H₂O₂ levels, highlighting a concentration-dependent trade-off. Palmitic acid was the predominant fatty acid under stress conditions. A strong positive correlation was observed between H₂O₂ concentration and total lipid content, indicating that exogenously induced oxidative stress can serve as a viable strategy to enhance lipid production in N. oceanica for biodiesel applications through a single-phase cultivation approach. microalgae Nannochloropsis oceanica peroxide hydrogen lipid biodiesel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Fatty acids are a major component of microalgal biomass, typically accounting for 5–50% of the cell’s dry weight (Simionato et al., 2013 ; Popko et al., 2016 ; Kiani et al., 2022 ). They are primarily found in the form of glycerolipids, which include phospholipids, glycolipids, and triacylglycerols (TAG). Among these, TAG-associated fatty acids are of particular commercial interest due to their applications in producing transportation fuels, bulk chemicals, nutraceuticals (e.g., ω-3 fatty acids), and food products (Chen et al., 2018 ; Halim et al., 2019 ; Oliver et al., 2020 ; Magoni et al., 2022 ). Microalgae offer a sustainable alternative to terrestrial crops for biodiesel and bulk product generation. They can grow in seawater, achieve higher areal productivity, and be cultivated in non-arable or offshore areas using photobioreactors, reducing dependency on agricultural land and freshwater resources (Bellou et al., 2014 ). Hence, microalgae-derived biofuels are classified as third-generation biofuels. Fatty acid content, lipid class distribution, and fatty acid chain characteristics vary significantly among microalgal species and are influenced by environmental conditions such as nutrient availability, temperature, pH, and light intensity [1,2]. Under favorable conditions, oleaginous microalgae produce low lipid levels, but stress—especially nutrient limitation—can stimulate lipid biosynthesis (Chisti, 2007 ; Georgianna & Mayfield, 2012 ; Ji et al., 2015 ). Nitrogen starvation, in particular, is highly effective, increasing TAG accumulation from below 2% up to 40% of dry weight (Yilancioglu et al., 2014). Stress-induced lipid accumulation is often accompanied by elevated antioxidant responses and intracellular reactive oxygen species (ROS) levels (Yang et al., 2013 ; Yilancioglu et al., 2014). In C. vulgaris , ROS levels are linked to neutral lipid content through an inverse and direct power law relationship (Menon et al., 2013 ). Exogenous ROS exposure—via agents like H₂O₂ or nanomaterials—can also trigger lipid biosynthesis (Yu et al., 2015 ). Studies have demonstrated that nitrogen depletion induces both ROS generation and lipid accumulation in diatoms and green microalgae (Liu et al., 2012 ; Li et al., 2011 ). High light intensity similarly raises ROS levels—including singlet oxygen (¹O₂), superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals—prompting the activation of antioxidant defenses such as glutathione peroxidase, superoxide dismutase, and non-enzymatic antioxidants like ascorbate, glutathione, carotenoids, and tocopherols (Hong et al., 2008 ). TAG synthesis, which requires more energy than carbohydrate synthesis, may serve to dissipate excess light energy and protect cells from photo-oxidative stress (Gordillo et al., 1998 ). While moderate ROS levels trigger adaptive lipid accumulation, excessive ROS can damage cells, leading to lipid consumption for energy homeostasis. Interestingly, antioxidant supplementation (e.g., sesamol, ginsenosides, ascorbic acid) has been shown to scavenge ROS and enhance polyunsaturated fatty acid (PUFA) content in lipid-producing microbes (Liu et al., 2015 ; Ren et al., 2017 ). This study focuses on evaluating the impact of various H₂O₂ concentrations on lipid accumulation and fatty acid profiles in Nannochloropsis oceanica , with the aim of optimizing its use as a biodiesel feedstock. Materials and methods Microalgal strain and culture condition N. oceanica CCAP 849/10 was obtained from the Culture Collection of Algae and Protozoa (CCAP), Scotland. Cultivation was carried out in 500 mL bubble-column photobioreactors containing f/2 medium (Guillard and Ryther, 1962 ), with continuous aeration using filtered air. The cultures were maintained at 25 ± 1°C under a light intensity of 100 µmol photons m⁻²s⁻¹, following a 14:10 hour light/dark photoperiod. To assess the impact of hydrogen peroxide (H₂O₂) on cell growth, biochemical composition, and lipid profile, H₂O₂ was added at varying concentrations (0, 100, 200, 300, and 400 µM) during the exponential growth phase. Cultures were incubated for 15 days post-treatment. Following incubation, biomass (based on cell number) and lipid content were measured. All experiments were conducted in triplicate to ensure reproducibility. Cell density Cell counts were performed with a hemocytometer to determine culture densities. The chlorophyll a content Chlorophyll a (Chl a) concentration was measured spectrophotometrically following the method described by Mackinney ( 1941 ). A 2 mL aliquot of the culture was collected and centrifuged at 3000 rpm for 10 minutes to pellet the cells. The supernatant was discarded, and the pellet was washed twice with distilled water to eliminate residual salts, each time followed by centrifugation. Subsequently, the washed cells were resuspended in 2 mL of 99.8% methanol and vigorously vortexed for 15 seconds to facilitate pigment extraction. After incubating for 20 minutes at room temperature, the suspension was centrifuged at 4000 rpm for 5 minutes. The absorbance of the resulting supernatant was then measured at 665 nm to determine chlorophyll a content. Fluorescence microscopy Nile Red (NR) staining was employed as a rapid and effective technique for visualizing and estimating the accumulation of neutral lipids with biodiesel potential in microalgal cells. Due to its strong lipophilic fluorescence properties, NR is widely used for lipid detection. For fluorescence microscopy, microalgal cells were initially fixed in 5% paraformaldehyde, followed by staining with a Nile Red stock solution (0.5 mg/mL, Sigma, USA). The stained samples were examined using an Olympus IX70 fluorescence microscope equipped with a 100× objective lens. Fluorescent images were captured using a cooled CCD camera, maintaining consistent exposure settings across all samples. The excitation and emission wavelengths were set to 460 ± 10 nm and 560–640 nm, respectively, to detect the characteristic yellow-gold fluorescence emitted by NR-bound neutral lipid bodies. Lipid content Algal biomass was harvested on day 15 by centrifugation at 4,000 rpm for 15 minutes. The resulting pellets were freeze-dried at − 46°C prior to lipid extraction. Total lipid content was extracted following a modified protocol of Bligh and Dyer ( 1959 ), using a chloroform–methanol mixture (2:1, v/v). For each extraction, 0.2 g of freeze-dried biomass was immersed in 50 mL of the solvent mixture and incubated for 24 hours. During this period, the samples were sonicated twice for 30 minutes at a frequency of 70 Hz to facilitate cell disruption. The suspension was subsequently filtered and washed twice with a potassium chloride (KCl) solution. The organic phase (lower layer) was collected in pre-weighed glass vials, and the solvent was evaporated at 40°C under reduced pressure. The lipid yield was calculated gravimetrically using the following formula: Y (%) = W L / W DA Where, W L is the weight of the extracted lipid and W DA is the dry algae biomass. Flow cytometric analysis for determination of lipid accumulation in cells To assess intracellular lipid accumulation, 5 µL of Nile Red stock solution (0.5 mg/mL) was added to 1 mL of cell suspension, following two washes with fresh growth medium. The stained suspension was gently vortexed and incubated in the dark at room temperature for 20 minutes. Nile Red fluorescence was measured using a flow cytometer equipped with a 488 nm argon laser. Upon excitation at this wavelength, Nile Red emits strong yellow-gold fluorescence in the presence of neutral lipids. Approximately 10,000 cells were analyzed per sample, with fluorescence intensity detected using logarithmic signal amplification. Unstained cells served as autofluorescence controls to ensure accurate detection of lipid-associated fluorescence. Transesterification and FAME analysis Total lipids were transesterified using 0.4 M KOH in methanol to produce fatty acid methyl esters (FAMEs), which were analyzed by gas chromatography (GC) with flame ionization detection (FID). The analysis was conducted on an Agilent HP6890A GC equipped with an Omega Wax 320 column (30 m × 0.32 mm I.D., 0.25 µm). Injector and detector temperatures were set at 260°C. The oven temperature program began at 60°C, increased to 170°C at 50°C min⁻¹, then to 180°C at 2°C min⁻¹ (held for 2 min), followed by increments to 230°C and 240°C at 2°C and 1°C min⁻¹, respectively. Helium served as the carrier gas at 30 mL min⁻¹. FAMEs were identified by comparing retention times to known standards (Sigma Chemical Co., USA), and their relative abundance was calculated using the normalization method. Catalase assay Total protein extraction was performed following the procedure outlined by Barbarino and Lourenço ( 2005 ), and protein concentration was quantified using the Bradford assay (Bradford, 1976 ). For the assessment of catalase (CAT) activity, 50 mg of algal biomass was homogenized in 2 mL of 0.5 M phosphate buffer (pH 7.5), followed by centrifugation at 12,000 rpm for 30 minutes at 4°C. The resulting supernatant was collected as the enzyme extract. CAT activity was measured spectrophotometrically by preparing a reaction mixture consisting of 1.6 mL phosphate buffer (pH 7.3), 100 µL of 3 mM EDTA, 200 µL of 0.3% H₂O₂, and 100 µL of the enzyme extract. The decrease in absorbance at 240 nm, corresponding to H₂O₂ decomposition, was recorded against a reagent blank lacking H₂O₂, as described by Aebi ( 1984 ). Data analysis One-way analysis of variance analyses (1-way ANOVA) were employed to assess the significance of lipid content variation between groups. When ANOVA confirmed significant variation, manifold comparisons among means value were done with Duncan’s test. SPSS v16 was used for statistical analyses. Results Growth analysis under different H 2 O 2 concentrations N. oceanica was cultured under varying concentrations of hydrogen peroxide to evaluate its growth response. As shown in Fig. 1 , both growth rate and biomass productivity declined progressively with increasing H₂O₂ concentrations, reaching up to 400 µM. A significant reduction in growth (p < 0.05) was observed after 14 days of exposure to 400 µM H₂O₂, compared to the untreated control group. Chlorophyll-a levels, used as an indirect indicator of algal growth, were measured to assess the impact of oxidative stress (Fig. 2 ). The microalga maintained stable growth at lower H₂O₂ concentrations (100 and 200 µM). However, exposure to higher levels of H₂O₂ resulted in notable suppression of chlorophyll-a content, reflecting impaired photosynthetic capacity and reduced biomass accumulation. Lipid accumulation analysis of N. oceanica under different H 2 O 2 concentrations Notably, the evaluation of lipid accumulation in N. oceanica under various concentrations of hydrogen peroxide indicated a concentration-dependent enhancement in lipid biosynthesis (Fig. 3 ). After 14 days of exposure to 400 µM H₂O₂, the lipid content showed a significant increase of 63% compared to the untreated control. These findings suggest that 400 µM H₂O₂ serves as an effective condition for promoting lipid overproduction in N. oceanica . The influence of H₂O₂-induced oxidative stress on fatty acid profiles is summarized in Table 4. The results demonstrated substantial alterations in fatty acid composition in response to increasing H₂O₂ concentrations. In control cultures, the predominant fatty acids included myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), and oleic acid (C18:1). Treatment with hydrogen peroxide resulted in elevated levels of palmitic acid and a general reduction in polyunsaturated fatty acids. Interestingly, the proportion of eicosapentaenoic acid (EPA), a valuable omega-3 fatty acid for human health, increased under H₂O₂ treatment (Table 1 ). Table 1 Fatty acid methyl ester (FAME) profile of N. oceanica cells cultivated under different H 2 O 2 concentrations. FAME (%) H 2 O 2 concentration (µM) 0 100 200 300 400 C12:0 n.d. n.d. 0.03 0.06 n.d. C14:0 6.13 4.37 4.23 3.88 4.39 C14:1 1.69 0.49 0.5 0.44 0.39 C15:0 n.d. 0.07 0.05 0.06 n.d. C16:0 31.78 39.03 40.15 39.02 39.78 C16:1 32.1 34.17 32.47 32.52 33.15 C16:2 n.d. 0.1 0.08 0.08 n.d. C17:0 n.d. 0.07 0.1 0.09 0.11 C17:1 n.d. 0.17 0.16 0.21 n.d. C18:0 3.17 1.37 1.78 1.52 1.26 C18:1 11.76 10.78 11.39 11.57 11.16 C18:2 1 1.01 0.11 0.6 0.95 C18:3 2.04 0.24 1.1 0.41 0.36 C20:0 1.1 0.35 0.25 0.15 0.5 C22:0 1.07 0.38 0.1 0.13 0.47 C20:5 1.9 2.6 2.63 2.22 3.04 C22:6 5.9 4.48 4.44 4.9 4.01 Others 0.46 0.07 0.08 0.14 0.14 Saturated 43.25 45.64 46.69 44.91 46.51 Monounsaturated 45.55 45.61 44.52 44.74 44.7 Polyunsaturated 10.84 8.43 8.36 8.36 8.36 Total 100.00 99.75 99.65 98.15 99.71 Fluorescent microscopy Cells stained with Nile red were examined under a fluorescence microscope. The micrographs revealed yellow-golden fluorescence indicative of neutral lipid bodies localized within the cytoplasm. These microscopic observations were consistent with the earlier quantitative findings, confirming enhanced lipid accumulation following H₂O₂ exposure. Specifically, treated cells exhibited a noticeable increase in both the size and number of cytoplasmic lipid droplets compared to the untreated control (Fig. 4 ). Flow cytometry Flow cytometric analysis of N. oceanica cultures treated with 400 µM H₂O₂, the condition yielding the highest lipid content, showed a uniform distribution of lipid-rich cells (Fig. 5 ). The results confirmed that oxidative stress induced by H₂O₂ led to an increase in intracellular lipid levels. Furthermore, flow cytometry revealed that this lipid accumulation was associated with elevated cellular granularity and increased biovolume, suggesting morphological and structural changes in response to oxidative stress (Fig. 5 ). Catalase activity under different H 2 O 2 concentrations Reactive oxygen species (ROS) accumulation in photosynthetic organisms is mitigated by an intrinsic antioxidant defense system, which includes key enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) [37]. To assess the intracellular oxidative stress status under hydrogen peroxide treatment, we focused on catalase (CAT) activity as a representative oxidative stress-responsive enzyme. Our results revealed a concentration-dependent increase in CAT activity in cultures exposed to elevated levels of H₂O₂ (Fig. 6 ), indicating activation of antioxidant defenses in response to exogenous oxidative stress. Discussion Lipid engineering in microalgae is achieved through conventional, genetic, and metabolic engineering approaches. One of the key advantages of utilizing microalgae as a biofuel feedstock lies in the flexibility to manipulate growth conditions to enhance biomass and lipid yields. Environmental and nutrient stresses—such as nitrogen limitation, temperature fluctuations, salinity, and heavy metal exposure—have been shown to upregulate stress-responsive proteins and promote lipid biosynthesis, particularly triacylglycerol (TAG) accumulation (Lei et al., 2012 ). Among these, oxidative stress has been strongly associated with enhanced TAG deposition in various microalgal species. In the present study, we investigated the impact of hydrogen peroxide (H₂O₂), an oxidative stress inducer, on biomass production and lipid accumulation in Nannochloropsis oceanica . Biomass productivity is a critical determinant for the commercial viability of microalgae-derived biodiesel. Our findings demonstrate that increasing H₂O₂ concentrations adversely affected the growth of N. oceanica , consistent with previous reports on the growth-inhibitory effects of H₂O₂ in algae (Zou et al., 2020 ). Similarly, Chen et al. ( 2019 ) reported complete inhibition of benthic cyanobacteria at 5 mg L⁻¹ H₂O₂. Chlorophyll-a, the principal photosynthetic pigment in microalgae, serves as an indicator of photosynthetic potential (Wang et al., 2016; Zhou et al., 2017). In this study, chlorophyll-a content declined significantly with increasing H₂O₂ concentrations, indicating suppressed chlorophyll biosynthesis and compromised photosynthetic efficiency in N. oceanica . Lipid content and productivity are fundamental metrics for evaluating microalgal suitability for biofuel production. Previous studies have shown that nutrient limitation can stimulate lipid accumulation in microalgae (Xin et al., 2010 ; Wu et al., 2014 ; Singh et al., 2015 ). Other environmental factors—such as elevated temperature, excessive light, and pH—may also induce lipid biosynthesis, potentially through reactive oxygen species (ROS)-mediated oxidative stress (Chen et al., 2011 ; Converti et al., 2009 ). In our study, Nile red fluorometric analysis revealed a concentration-dependent increase in lipid accumulation in response to H₂O₂ treatment. This suggests that inducing oxidative stress at the stationary growth phase, following cultivation under optimal conditions, may represent a cost-effective strategy for maximizing lipid yields. Compared to nitrogen starvation—which reduces biomass yield—this method may offer a more balanced approach to biofuel production. Our findings support previous work indicating that H₂O₂-induced oxidative stress enhances neutral lipid accumulation, as observed in Chlorella sorokiniana C3 (Zhang et al., 2013 ). These results highlight the potential of exogenous oxidative stress as a practical tool for improving lipid productivity in microalgae. Conclusions In this study, we presented evidence supporting that H 2 O 2 causes oxidative stress and lipid accumulation. In addition, we showed that oxidative stress by itself can cause lipid accumulation, these observations are helpful for utilization of Nannochloropsis oceanica for biodiesel production. 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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-6458745","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451659309,"identity":"ed7473de-06e0-4479-93ac-9baeb1cf6b2a","order_by":0,"name":"Majid Mahdieh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYJACZgY2CQYGCeYDQHYCRCiBOC1sCSRpAZISPAZEqAUC3QbuxM8FZRaJ/dI9Hz9X1KQlNrAffsDwcA9uLWYHeDdLzzgnkThzztnNkmeO5SQ28KQZMCQ8w6Pl/tsN0rxtEokbbuRukGxgq0hsYMgBuu8Aflt+g7Tsv5Hz+GfDP6AW/jcEtWyD2CKRwybZ2AZ0mARhW7ZZ85yTMJ5xI83MsrEvzbhN4pnBAUIOu81TVifbPyP58c2Gb8my/fzJDx/+wKMFBhwbYCxQHBGhgYHBnhhFo2AUjIJRMEIBADfvU8V++8qWAAAAAElFTkSuQmCC","orcid":"","institution":"Arak University","correspondingAuthor":true,"prefix":"","firstName":"Majid","middleName":"","lastName":"Mahdieh","suffix":""},{"id":451659310,"identity":"3659bea8-7bc6-4429-9db8-884ceb96a94d","order_by":1,"name":"Abolfazl Taghavi","email":"","orcid":"","institution":"Arak University","correspondingAuthor":false,"prefix":"","firstName":"Abolfazl","middleName":"","lastName":"Taghavi","suffix":""},{"id":451659311,"identity":"92a1ac67-2b97-404d-85d5-dd333c5bf6cd","order_by":2,"name":"Mona Nezarat","email":"","orcid":"","institution":"Arak University","correspondingAuthor":false,"prefix":"","firstName":"Mona","middleName":"","lastName":"Nezarat","suffix":""}],"badges":[],"createdAt":"2025-04-16 02:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6458745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6458745/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82001821,"identity":"1f71ae47-127e-4410-99f8-880020f01525","added_by":"auto","created_at":"2025-05-05 20:01:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65747,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations on the (A) growth and (B) biomass productivity of \u003cem\u003eN. oceanica.\u003c/em\u003e The error bars correspond to ±1 SD of triplicate measurements. The value shown with different letters are significantly different (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/e2c9af38d9b7b805e713aa97.jpg"},{"id":82001823,"identity":"e23b65e4-7755-4786-b575-b524755a174f","added_by":"auto","created_at":"2025-05-05 20:01:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75136,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations on chlorophyll a content (mg ml\u003csup\u003e-1\u003c/sup\u003e) of \u003cem\u003eN. oceanica.\u003c/em\u003e The error bars correspond to ±1 SD of triplicate measurements. The value shown with different letters are significantly different (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/5bc442d898bd54d847794dc9.jpg"},{"id":82001822,"identity":"c1e7693b-1dc4-45b3-810d-96d427beaee2","added_by":"auto","created_at":"2025-05-05 20:01:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92433,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations on the lipid productivity of \u003cem\u003eN. oceanica.\u003c/em\u003e The error bars correspond to ±1 SD of triplicate measurements. The value shown with different letters are significantly different (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/78ddb3d81e6e14e1ba16b978.jpg"},{"id":82002182,"identity":"21c5c20c-592f-4014-99b1-85368bd8d22e","added_by":"auto","created_at":"2025-05-05 20:09:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33589,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microphotographs of \u003cem\u003e\u003cstrong\u003eN. oceanica\u003c/strong\u003e\u003c/em\u003e stained with Nile-Red fluorescence dye and screened under 400X magnification. A) Control group, 0 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration cultivation condition. B) 100 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, C) 200 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, D) 300 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and E) 400 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/69481fa5573c3b134453417f.jpg"},{"id":82001829,"identity":"b5247992-d815-43bd-beff-7606b9058085","added_by":"auto","created_at":"2025-05-05 20:01:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21041,"visible":true,"origin":"","legend":"\u003cp\u003eThe flowmetric analysis of Nile red-stained cells.\u003cstrong\u003e \u003c/strong\u003eSSC (Side Scatter) and FSC (Forward Scatter) expressing cellular granulation and cellular size, respectively, under 400 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/5391cc431aa649b779533d27.jpg"},{"id":82001830,"identity":"3450f5a4-9f8f-4eaf-b3a2-6420621aa816","added_by":"auto","created_at":"2025-05-05 20:01:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103536,"visible":true,"origin":"","legend":"\u003cp\u003eCatalase activity under different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations. Spectrophotometric enzymatic assay for catalase (CAT). Data demonstrate the mean values of triplicates ±SE.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/10c749aa148db9959280fa75.jpg"},{"id":94187095,"identity":"2c9055f6-a576-47b7-a6be-bc99e8d8a48b","added_by":"auto","created_at":"2025-10-23 11:08:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135127,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6458745/v1/6a3eca6b-9645-48e5-a9d1-f6441be8435e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oxidative stress enhances lipid production and improves fatty acid composition for biodiesel production in microalga Nannochloropsis oceanica","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFatty acids are a major component of microalgal biomass, typically accounting for 5\u0026ndash;50% of the cell\u0026rsquo;s dry weight (Simionato et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Popko et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kiani et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). They are primarily found in the form of glycerolipids, which include phospholipids, glycolipids, and triacylglycerols (TAG). Among these, TAG-associated fatty acids are of particular commercial interest due to their applications in producing transportation fuels, bulk chemicals, nutraceuticals (e.g., ω-3 fatty acids), and food products (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Halim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oliver et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Magoni et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroalgae offer a sustainable alternative to terrestrial crops for biodiesel and bulk product generation. They can grow in seawater, achieve higher areal productivity, and be cultivated in non-arable or offshore areas using photobioreactors, reducing dependency on agricultural land and freshwater resources (Bellou et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Hence, microalgae-derived biofuels are classified as third-generation biofuels.\u003c/p\u003e \u003cp\u003eFatty acid content, lipid class distribution, and fatty acid chain characteristics vary significantly among microalgal species and are influenced by environmental conditions such as nutrient availability, temperature, pH, and light intensity [1,2]. Under favorable conditions, oleaginous microalgae produce low lipid levels, but stress\u0026mdash;especially nutrient limitation\u0026mdash;can stimulate lipid biosynthesis (Chisti, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Georgianna \u0026amp; Mayfield, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ji et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nitrogen starvation, in particular, is highly effective, increasing TAG accumulation from below 2% up to 40% of dry weight (Yilancioglu et al., 2014).\u003c/p\u003e \u003cp\u003eStress-induced lipid accumulation is often accompanied by elevated antioxidant responses and intracellular reactive oxygen species (ROS) levels (Yang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yilancioglu et al., 2014). In \u003cem\u003eC. vulgaris\u003c/em\u003e, ROS levels are linked to neutral lipid content through an inverse and direct power law relationship (Menon et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Exogenous ROS exposure\u0026mdash;via agents like H₂O₂ or nanomaterials\u0026mdash;can also trigger lipid biosynthesis (Yu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStudies have demonstrated that nitrogen depletion induces both ROS generation and lipid accumulation in diatoms and green microalgae (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). High light intensity similarly raises ROS levels\u0026mdash;including singlet oxygen (\u0026sup1;O₂), superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals\u0026mdash;prompting the activation of antioxidant defenses such as glutathione peroxidase, superoxide dismutase, and non-enzymatic antioxidants like ascorbate, glutathione, carotenoids, and tocopherols (Hong et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). TAG synthesis, which requires more energy than carbohydrate synthesis, may serve to dissipate excess light energy and protect cells from photo-oxidative stress (Gordillo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile moderate ROS levels trigger adaptive lipid accumulation, excessive ROS can damage cells, leading to lipid consumption for energy homeostasis. Interestingly, antioxidant supplementation (e.g., sesamol, ginsenosides, ascorbic acid) has been shown to scavenge ROS and enhance polyunsaturated fatty acid (PUFA) content in lipid-producing microbes (Liu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study focuses on evaluating the impact of various H₂O₂ concentrations on lipid accumulation and fatty acid profiles in \u003cem\u003eNannochloropsis oceanica\u003c/em\u003e, with the aim of optimizing its use as a biodiesel feedstock.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroalgal strain and culture condition\u003c/h2\u003e \u003cp\u003e \u003cem\u003eN. oceanica\u003c/em\u003e CCAP 849/10 was obtained from the Culture Collection of Algae and Protozoa (CCAP), Scotland. Cultivation was carried out in 500 mL bubble-column photobioreactors containing f/2 medium (Guillard and Ryther, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1962\u003c/span\u003e), with continuous aeration using filtered air. The cultures were maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a light intensity of 100 \u0026micro;mol photons m⁻\u0026sup2;s⁻\u0026sup1;, following a 14:10 hour light/dark photoperiod.\u003c/p\u003e \u003cp\u003eTo assess the impact of hydrogen peroxide (H₂O₂) on cell growth, biochemical composition, and lipid profile, H₂O₂ was added at varying concentrations (0, 100, 200, 300, and 400 \u0026micro;M) during the exponential growth phase. Cultures were incubated for 15 days post-treatment.\u003c/p\u003e \u003cp\u003eFollowing incubation, biomass (based on cell number) and lipid content were measured. All experiments were conducted in triplicate to ensure reproducibility.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell density\u003c/h3\u003e\n\u003cp\u003eCell counts were performed with a hemocytometer to determine culture densities.\u003c/p\u003e\n\u003ch3\u003eThe chlorophyll a content\u003c/h3\u003e\n\u003cp\u003eChlorophyll a (Chl a) concentration was measured spectrophotometrically following the method described by Mackinney (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1941\u003c/span\u003e). A 2 mL aliquot of the culture was collected and centrifuged at 3000 rpm for 10 minutes to pellet the cells. The supernatant was discarded, and the pellet was washed twice with distilled water to eliminate residual salts, each time followed by centrifugation. Subsequently, the washed cells were resuspended in 2 mL of 99.8% methanol and vigorously vortexed for 15 seconds to facilitate pigment extraction. After incubating for 20 minutes at room temperature, the suspension was centrifuged at 4000 rpm for 5 minutes. The absorbance of the resulting supernatant was then measured at 665 nm to determine chlorophyll a content.\u003c/p\u003e\n\u003ch3\u003eFluorescence microscopy\u003c/h3\u003e\n\u003cp\u003eNile Red (NR) staining was employed as a rapid and effective technique for visualizing and estimating the accumulation of neutral lipids with biodiesel potential in microalgal cells. Due to its strong lipophilic fluorescence properties, NR is widely used for lipid detection. For fluorescence microscopy, microalgal cells were initially fixed in 5% paraformaldehyde, followed by staining with a Nile Red stock solution (0.5 mg/mL, Sigma, USA). The stained samples were examined using an Olympus IX70 fluorescence microscope equipped with a 100\u0026times; objective lens. Fluorescent images were captured using a cooled CCD camera, maintaining consistent exposure settings across all samples. The excitation and emission wavelengths were set to 460\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm and 560\u0026ndash;640 nm, respectively, to detect the characteristic yellow-gold fluorescence emitted by NR-bound neutral lipid bodies.\u003c/p\u003e\n\u003ch3\u003eLipid content\u003c/h3\u003e\n\u003cp\u003eAlgal biomass was harvested on day 15 by centrifugation at 4,000 rpm for 15 minutes. The resulting pellets were freeze-dried at \u0026minus;\u0026thinsp;46\u0026deg;C prior to lipid extraction. Total lipid content was extracted following a modified protocol of Bligh and Dyer (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1959\u003c/span\u003e), using a chloroform\u0026ndash;methanol mixture (2:1, v/v). For each extraction, 0.2 g of freeze-dried biomass was immersed in 50 mL of the solvent mixture and incubated for 24 hours. During this period, the samples were sonicated twice for 30 minutes at a frequency of 70 Hz to facilitate cell disruption. The suspension was subsequently filtered and washed twice with a potassium chloride (KCl) solution. The organic phase (lower layer) was collected in pre-weighed glass vials, and the solvent was evaporated at 40\u0026deg;C under reduced pressure. The lipid yield was calculated gravimetrically using the following formula:\u003c/p\u003e \u003cp\u003e \u003cem\u003eY\u003c/em\u003e(%)\u0026thinsp;=\u0026thinsp;\u003cem\u003eW\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e/\u003cem\u003eW\u003c/em\u003e\u003csub\u003eDA\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eWhere, \u003cem\u003eW\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e is the weight of the extracted lipid and \u003cem\u003eW\u003c/em\u003e\u003csub\u003eDA\u003c/sub\u003e is the dry algae biomass.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis for determination of lipid accumulation in cells\u003c/h2\u003e \u003cp\u003eTo assess intracellular lipid accumulation, 5 \u0026micro;L of Nile Red stock solution (0.5 mg/mL) was added to 1 mL of cell suspension, following two washes with fresh growth medium. The stained suspension was gently vortexed and incubated in the dark at room temperature for 20 minutes. Nile Red fluorescence was measured using a flow cytometer equipped with a 488 nm argon laser. Upon excitation at this wavelength, Nile Red emits strong yellow-gold fluorescence in the presence of neutral lipids. Approximately 10,000 cells were analyzed per sample, with fluorescence intensity detected using logarithmic signal amplification. Unstained cells served as autofluorescence controls to ensure accurate detection of lipid-associated fluorescence.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransesterification and FAME analysis\u003c/h3\u003e\n\u003cp\u003eTotal lipids were transesterified using 0.4 M KOH in methanol to produce fatty acid methyl esters (FAMEs), which were analyzed by gas chromatography (GC) with flame ionization detection (FID). The analysis was conducted on an Agilent HP6890A GC equipped with an Omega Wax 320 column (30 m \u0026times; 0.32 mm I.D., 0.25 \u0026micro;m). Injector and detector temperatures were set at 260\u0026deg;C. The oven temperature program began at 60\u0026deg;C, increased to 170\u0026deg;C at 50\u0026deg;C min⁻\u0026sup1;, then to 180\u0026deg;C at 2\u0026deg;C min⁻\u0026sup1; (held for 2 min), followed by increments to 230\u0026deg;C and 240\u0026deg;C at 2\u0026deg;C and 1\u0026deg;C min⁻\u0026sup1;, respectively. Helium served as the carrier gas at 30 mL min⁻\u0026sup1;. FAMEs were identified by comparing retention times to known standards (Sigma Chemical Co., USA), and their relative abundance was calculated using the normalization method.\u003c/p\u003e\n\u003ch3\u003eCatalase assay\u003c/h3\u003e\n\u003cp\u003eTotal protein extraction was performed following the procedure outlined by Barbarino and Louren\u0026ccedil;o (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and protein concentration was quantified using the Bradford assay (Bradford, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). For the assessment of catalase (CAT) activity, 50 mg of algal biomass was homogenized in 2 mL of 0.5 M phosphate buffer (pH 7.5), followed by centrifugation at 12,000 rpm for 30 minutes at 4\u0026deg;C. The resulting supernatant was collected as the enzyme extract. CAT activity was measured spectrophotometrically by preparing a reaction mixture consisting of 1.6 mL phosphate buffer (pH 7.3), 100 \u0026micro;L of 3 mM EDTA, 200 \u0026micro;L of 0.3% H₂O₂, and 100 \u0026micro;L of the enzyme extract. The decrease in absorbance at 240 nm, corresponding to H₂O₂ decomposition, was recorded against a reagent blank lacking H₂O₂, as described by Aebi (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1984\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eOne-way analysis of variance analyses (1-way ANOVA) were employed to assess the significance of lipid content variation between groups. When ANOVA confirmed significant variation, manifold comparisons among means value were done with Duncan\u0026rsquo;s test. SPSS v16 was used for statistical analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGrowth analysis under different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations\u003c/h2\u003e \u003cp\u003e \u003cem\u003eN. oceanica\u003c/em\u003e was cultured under varying concentrations of hydrogen peroxide to evaluate its growth response. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, both growth rate and biomass productivity declined progressively with increasing H₂O₂ concentrations, reaching up to 400 \u0026micro;M. A significant reduction in growth (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed after 14 days of exposure to 400 \u0026micro;M H₂O₂, compared to the untreated control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChlorophyll-a levels, used as an indirect indicator of algal growth, were measured to assess the impact of oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The microalga maintained stable growth at lower H₂O₂ concentrations (100 and 200 \u0026micro;M). However, exposure to higher levels of H₂O₂ resulted in notable suppression of chlorophyll-a content, reflecting impaired photosynthetic capacity and reduced biomass accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLipid accumulation analysis of\u003c/b\u003e \u003cb\u003eN. oceanica\u003c/b\u003e \u003cb\u003eunder different H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003econcentrations\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNotably, the evaluation of lipid accumulation in \u003cem\u003eN. oceanica\u003c/em\u003e under various concentrations of hydrogen peroxide indicated a concentration-dependent enhancement in lipid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After 14 days of exposure to 400 \u0026micro;M H₂O₂, the lipid content showed a significant increase of 63% compared to the untreated control. These findings suggest that 400 \u0026micro;M H₂O₂ serves as an effective condition for promoting lipid overproduction in \u003cem\u003eN. oceanica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe influence of H₂O₂-induced oxidative stress on fatty acid profiles is summarized in Table\u0026nbsp;4. The results demonstrated substantial alterations in fatty acid composition in response to increasing H₂O₂ concentrations. In control cultures, the predominant fatty acids included myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), and oleic acid (C18:1). Treatment with hydrogen peroxide resulted in elevated levels of palmitic acid and a general reduction in polyunsaturated fatty acids. Interestingly, the proportion of eicosapentaenoic acid (EPA), a valuable omega-3 fatty acid for human health, increased under H₂O₂ treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eFatty acid methyl ester (FAME) profile of \u003cem\u003eN. oceanica\u003c/em\u003e cells cultivated under different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations.\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=\"left\" 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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFAME (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC12:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC15:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC17:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC17:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC20:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC22:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC20:5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC22:6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaturated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e44.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonounsaturated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e44.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyunsaturated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.71\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=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent microscopy\u003c/h2\u003e \u003cp\u003eCells stained with Nile red were examined under a fluorescence microscope. The micrographs revealed yellow-golden fluorescence indicative of neutral lipid bodies localized within the cytoplasm. These microscopic observations were consistent with the earlier quantitative findings, confirming enhanced lipid accumulation following H₂O₂ exposure. Specifically, treated cells exhibited a noticeable increase in both the size and number of cytoplasmic lipid droplets compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometric analysis of \u003cem\u003eN. oceanica\u003c/em\u003e cultures treated with 400 \u0026micro;M H₂O₂, the condition yielding the highest lipid content, showed a uniform distribution of lipid-rich cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results confirmed that oxidative stress induced by H₂O₂ led to an increase in intracellular lipid levels. Furthermore, flow cytometry revealed that this lipid accumulation was associated with elevated cellular granularity and increased biovolume, suggesting morphological and structural changes in response to oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCatalase activity under different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations\u003c/h2\u003e \u003cp\u003eReactive oxygen species (ROS) accumulation in photosynthetic organisms is mitigated by an intrinsic antioxidant defense system, which includes key enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) [37]. To assess the intracellular oxidative stress status under hydrogen peroxide treatment, we focused on catalase (CAT) activity as a representative oxidative stress-responsive enzyme. Our results revealed a concentration-dependent increase in CAT activity in cultures exposed to elevated levels of H₂O₂ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating activation of antioxidant defenses in response to exogenous oxidative stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLipid engineering in microalgae is achieved through conventional, genetic, and metabolic engineering approaches. One of the key advantages of utilizing microalgae as a biofuel feedstock lies in the flexibility to manipulate growth conditions to enhance biomass and lipid yields. Environmental and nutrient stresses\u0026mdash;such as nitrogen limitation, temperature fluctuations, salinity, and heavy metal exposure\u0026mdash;have been shown to upregulate stress-responsive proteins and promote lipid biosynthesis, particularly triacylglycerol (TAG) accumulation (Lei et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Among these, oxidative stress has been strongly associated with enhanced TAG deposition in various microalgal species.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the impact of hydrogen peroxide (H₂O₂), an oxidative stress inducer, on biomass production and lipid accumulation in \u003cem\u003eNannochloropsis oceanica\u003c/em\u003e. Biomass productivity is a critical determinant for the commercial viability of microalgae-derived biodiesel. Our findings demonstrate that increasing H₂O₂ concentrations adversely affected the growth of \u003cem\u003eN. oceanica\u003c/em\u003e, consistent with previous reports on the growth-inhibitory effects of H₂O₂ in algae (Zou et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, Chen et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported complete inhibition of benthic cyanobacteria at 5 mg L⁻\u0026sup1; H₂O₂.\u003c/p\u003e \u003cp\u003eChlorophyll-a, the principal photosynthetic pigment in microalgae, serves as an indicator of photosynthetic potential (Wang et al., 2016; Zhou et al., 2017). In this study, chlorophyll-a content declined significantly with increasing H₂O₂ concentrations, indicating suppressed chlorophyll biosynthesis and compromised photosynthetic efficiency in \u003cem\u003eN. oceanica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eLipid content and productivity are fundamental metrics for evaluating microalgal suitability for biofuel production. Previous studies have shown that nutrient limitation can stimulate lipid accumulation in microalgae (Xin et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Other environmental factors\u0026mdash;such as elevated temperature, excessive light, and pH\u0026mdash;may also induce lipid biosynthesis, potentially through reactive oxygen species (ROS)-mediated oxidative stress (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Converti et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, Nile red fluorometric analysis revealed a concentration-dependent increase in lipid accumulation in response to H₂O₂ treatment. This suggests that inducing oxidative stress at the stationary growth phase, following cultivation under optimal conditions, may represent a cost-effective strategy for maximizing lipid yields. Compared to nitrogen starvation\u0026mdash;which reduces biomass yield\u0026mdash;this method may offer a more balanced approach to biofuel production.\u003c/p\u003e \u003cp\u003eOur findings support previous work indicating that H₂O₂-induced oxidative stress enhances neutral lipid accumulation, as observed in \u003cem\u003eChlorella sorokiniana\u003c/em\u003e C3 (Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These results highlight the potential of exogenous oxidative stress as a practical tool for improving lipid productivity in microalgae.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we presented evidence supporting that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e causes oxidative stress and lipid accumulation. In addition, we showed that oxidative stress by itself can cause lipid accumulation, these observations are helpful for utilization of \u003cem\u003eNannochloropsis oceanica\u003c/em\u003e for biodiesel production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Research and Technology Deputy of Arak University.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.M supervised research and wrote the main manuscript. A.T. and M.N. did experiments. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAebi H (1984) Catalase in vitro. 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PLoS One 8:e69225.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"microalgae, Nannochloropsis oceanica, peroxide hydrogen, lipid, biodiesel","lastPublishedDoi":"10.21203/rs.3.rs-6458745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6458745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe depletion of fossil fuel reserves, escalating energy prices, rising global energy demand, and heightened awareness of climate change have accelerated efforts to develop sustainable biofuel alternatives. Microalgae are considered a promising candidate for biodiesel production due to their rapid biomass accumulation, high lipid content, and superior photosynthetic efficiency compared to conventional terrestrial crops. This study examined the effect of oxidative stress, induced by hydrogen peroxide (H₂O₂), on the growth and lipid productivity of the marine microalga \u003cem\u003eNannochloropsis oceanica\u003c/em\u003e. Five concentrations of H₂O₂ (0, 100, 200, 300, and 400 \u0026micro;M) were applied during the exponential growth phase. Cultures were maintained under controlled conditions, and at the end of the cultivation period, biomass was harvested and freeze-dried. Lipids were extracted using a modified Bligh and Dyer protocol, and fatty acid profiles were determined through gas chromatography. The results demonstrated a significant enhancement in lipid accumulation under oxidative stress, with the highest lipid content\u0026mdash;a 64% increase over the control\u0026mdash;recorded at 400 \u0026micro;M H₂O₂. However, biomass productivity decreased at elevated H₂O₂ levels, highlighting a concentration-dependent trade-off. Palmitic acid was the predominant fatty acid under stress conditions. A strong positive correlation was observed between H₂O₂ concentration and total lipid content, indicating that exogenously induced oxidative stress can serve as a viable strategy to enhance lipid production in \u003cem\u003eN. oceanica\u003c/em\u003e for biodiesel applications through a single-phase cultivation approach.\u003c/p\u003e","manuscriptTitle":"Oxidative stress enhances lipid production and improves fatty acid composition for biodiesel production in microalga Nannochloropsis oceanica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 20:00:55","doi":"10.21203/rs.3.rs-6458745/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa2c6bc3-14aa-44a5-aee3-227799d517bd","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-23T11:08:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 20:00:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6458745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6458745","identity":"rs-6458745","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}