Strategies for carbon regulation to enhance the efficient enrichment of energy storage materials in Tribonema minus

Strategies for carbon regulation to enhance the efficient enrichment of energy storage materials in Tribonema minus | 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 Strategies for carbon regulation to enhance the efficient enrichment of energy storage materials in Tribonema minus luyun Cai, min Zhao, yuchuan Bian, bingbing Dong, ran Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7739724/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Tribonema minus efficiently accumulates energy storage substances such as lipids and polysaccharides, demonstrating potential for bioenergy and high-value-added product development. Carbon source regulation is a key strategy to promote energy storage compound enrichment. This study evaluated how different amounts of glycerol and glucose affected the carbon dioxide, lipid composition, photosynthesis rate, and metabolic pathways of Tribonema minus . The results showed that as glycerol and glucose concentrations increased, the biomass and lipid yield of each treatment group increased linearly, Among them, 0.4 g L − 1 glycerol treatment markedly raised the lipid content (48.1%) and biomass (1.81 g L − 1 ), while 0.5 g L − 1 glucose treatment showed a more significant promoting effect, and increased biomass 3.06-fold and lipid content 1.27-fold ver. control. In addition, glycerol and glucose significantly increased the protein content of microalgae, with 0.4 g L − 1 glycerol yielding the highest protein content (900.91 mg L − 1 ). Although photosynthetic efficiency decreased in the late culture stage of each treatment group, the photochemical efficiency (Fv/Fm) remained above 0.6 at all times. Non-targeted metabolomics analysis showed that the addition of 0.4 g L − 1 glycerol and 0.5 g L − 1 glucose significantly promoted fatty acid synthesis by enhancing glycolysis and providing sufficient glycerol-3-phosphate precursors, emphasizing the positive role of these carbon sources in lipid accumulation. The results indicate that glycerol and glucose serve as excellent carbon sources for T. minus culture, facilitating a cost-efficient method to enhance biomass and lipid yields for biofuel production. Carbon source regulation Lipid accumulation Non-targeted metabolomics Tribonema minus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Microalgae, as highly efficient photosynthetic autotrophic organisms, demonstrate significant promise in the advancement of energy from renewable sources as well as high-value-added products. Compared with traditional terrestrial crops, microalgae grow rapidly, produce high biomass yields, avoid using agricultural land and can employ wastewater and waste gas for cultivation substrates. (Bumbac et al. 2024 ). Under carbon source regulation, microalgae can significantly enhance the accumulation of storage substances such as lipids (up to over 50% of dry cell weight) and polysaccharides (20–60%) (Song et al. 2023 ). These products can be further converted into biodiesel and aviation fuel with higher cetane numbers and better combustion performance (Arora et al. 2017 ; Neag et al. 2023 ), highlighting the synthetic potential of microalgae under carbon source guidance. Some microalgae (such as Tribonema minus ) possess an active glyoxylic acid cycle pathway, enabling them to efficiently utilize organic carbon sources and demonstrating significant carbon metabolism plasticity. Notably, Tribonema minus (a filamentous microalga of the Chrysophyta phylum) has emerged as a promising candidate for bioenergy and high-value chemical production due to its unique photosynthetic physiology and high energy storage capacity. Physiological studies demonstrate that T. minus achieves lipid contents of 45–55% of dry cell weight under nitrogen stress, with > 80% of fatty acids comprising C16–C18 chains — an ideal profile for biodiesel (Feng et al. 2019 ). Critically, certain strains simultaneously accumulate triacylglycerol (TAG) and other high-value products, enabling combined energy and functional compound production in a single cultivation process (Davis et al. 2021 ). These attributes render T. minus ideal for microalgal biorefining. Carbon metabolism regulation in T. minus reveals remarkable plasticity. Under autotrophic conditions, polysaccharides dominate accumulation (35–40% of dry weight), whereas sodium acetate supplementation in mixotrophic cultures increases lipid content to > 50% without compromising growth rate (Dong et al. 2025 ). This adaptability stems from a unique metabolic network: metabolomic analyses confirm a functional glyoxylate cycle that directly converts acetate into lipid precursors (Wang et al. 2018 ). Carbon source regulation is a key factor controlling microalgal growth and metabolic flux during energy storage compound biosynthesis (Oliveira et al. 2021 ). Although microalgae primarily utilize CO₂ through photosynthesis for growth and energy storage under natural conditions (Xie et al. 2022 ), inorganic carbon sources are often constrained by environmental CO₂ concentration, light intensity, and nutrient availability, limiting efficient compound enrichment (Luo. 2020). Consequently, recent studies focus on organic carbon supplementation (e.g glucose, glycerol, acetate) to redirect metabolic networks toward lipid and polysaccharide synthesis (Gao et al. 2022 ; Li et al. 2025 ). These compounds provide direct carbon skeletons and energy while indirectly modulating carbon flux distribution, reducing equivalent generation, and signaling pathways to enhance storage efficiency (Sun et al. 2020 ; Huang et al. 2021 ). For instance, glucose (an easily assimilated monosaccharide) significantly boosts biomass and lipid content in many microalgae, whereas glycerol uniquely promotes lipid synthesis and osmotic regulation (Yu et al. 2023 ). Carbon source type and concentration further influence trophic modes (autotrophy, heterotrophy, mixotrophy), photosynthetic activity, and cellular component allocation, ultimately determining biomass and metabolite yields (Mondal et al. 2017 ). Despite this progress, carbon source regulation mechanisms in T. minus remain poorly understood, particularly regarding systematic optimization of energy storage compound enrichment and metabolic pathway modulation by organic carbon sources. Advances in metabolomics and transcriptomics now enable elucidation of molecular mechanisms underlying metabolic network remodeling and functional molecule accumulation in T. minus under carbon source regulation-a pivotal scientific challenge in this field (Long. 2017). 2 Materials and Methods 2.1 Algal culture Tribonema minus is from the Ningbo Science and Technology Innovation Center of Zhejiang University and is currently stored in the algae seed room of the Food Branch. Algal cells were cultured in 1 L flasks with starting infection of 0.3 g L − 1 and irradiated at 26 ± 1°C with a 90 ± 5 µmol m − 2 s − 1 intensity LED lamp, shaking 3 times a day to ensure uniform distribution. Algal cells cultivated to the exponential phase were seeded in Bold base medium (BBM) supplemented by varying quantities of glycerol and glucose at (0.2, 0.4, 0.6, 0.8 and 1.0 g L − 1 ) and glucose at (0.2, 0.3, 0.4, 0.5 and 0.6 g L − 1 ) with other nutrient components unchanged: NaNO₃ 0.3 g L − 1 , MgSO₄·7H₂O 0.08 g L − 1 , NaCl 0.03 g L − 1 , K₂HPO₄ 0.3 g L − 1 , KH₂PO₄ 0.18 g L − 1 , CaCl₂·2H₂O 0.03 g L − 1 , H₃BO₃ 0.01 g L − 1 , EDTA-Na₂ 0.05 g L − 1 , KOH 0.03 g L − 1 , ZnSO₄·7H₂O 8.82 mg L − 1 , Co(NO₃)₂·6H₂O 0.49 mg L − 1 , MnCl₂·4H₂O 1.44 mg L − 1 , CuSO₄·5H₂O 1.57 mg L − 1 , Na₂MoO₄·2H₂O 1.2 mg L − 1 , FeSO₄·7H₂O 4.98 mg L − 1 . Cultures were incubated under LED illumination (90 µmol m − 2 s − 1 ) with a 12 h:12 h light:dark photoperiod at 26 ± 1°C. Continuous aeration was provided via compressed air, and cultures were shaken manually three times daily. After 14 days of cultivation, algal cells grown in carbon-free medium served as the control group (CK). Samples were collected bi-daily for the study of carbon dioxide, total lipids, proteins, carbohydrates, and photosynthetic pigments. 2.2 Assessment of biomass and total lipid content Biomass concentration was determined gravimetrically. iberglass screens with pore sizes of 0.45 µm were dried at 105°C for 12 h, desiccated to constant weight (DW₁), and reweighed. A 10 mL algal suspension (V) was passed through filters, subjected to three washes with deionized water, dried at 105°C for 12 hours, and allowed to cool to a constant weight (DW₂). Biomass concentration was calculated as: $$\:\text{DW}\left(\text{g}{\text{L}}^{\text{-1}}\right)\text{=}\frac{\text{(}{\text{DW}}_{\text{2}}\text{-}{\text{DW}}_{\text{1}}\text{)}}{\text{V}}$$ 1 Calculate the biomass yield P (g L − 1 d − 1 ) utilizing the equation provided below y. $$\:\text{P}\text{=}\frac{\text{(}{\text{X}}_{\text{t}}\text{-}{\text{X}}_{\text{0}}\text{)}}{\text{t}}\text{}$$ 2 X 0 and X t denote the amount of biomass (g L − 1 ) after the initial and t days, respectively; t denotes the total incubation duration. The total lipid content of Tribonema minus was determined by vanillin phosphate chromogenic method, and the filtered algae waste was rinsed twice with ionized water, then dried in a freeze dryer for 72 h, and the lyophilized algae powder was stored in a freeze − 80°C refrigerator. In order to facilitate lipid extraction, fifty milligrams of algae powder were dissolved in a designated volume of deionized water, Subsequently, 2 ml of sulfuric acid were introduced. The solution was subjected to heating in a water bath at 90°C for a duration of 20 minutes. then allowed to cool to room temperature. Subsequently, 3 mL of vanillin phosphate chromogenic reagent was incorporated, thoroughly mixed, and allowed to develop at room temperature for 20 minutes. After then, the amount of absorption was taken at 530 nm, and the lipid content was calculated in comparison to the standard curve (Gao et al. 2019 ; Pinger et al. 2022 ). The lipid content is quantified as a percentage of dry biomass. Standard Curve: y = 1.423X + 0.025 R 2 = 0.998 (3) Lipid production P (g L − 1 d − 1 ) is determined as follows: $$\:{\text{Ρ}}_{\text{lipid}}\text{=}\text{}\frac{{\text{DW}}_{\text{x}}\text{×}{\text{C}}_{\text{x}}\text{-}{\text{DW}}_{\text{1}}\text{×}{\text{C}}_{\text{1}}}{\text{t}}$$ 4 where lipid productivity (g L − 1 d − 1 ) is indicated; DW represents microalgae biomass (g L − 1 ); Indicates lipid content (%); DW 1 and DW x represent the biomass of microalgae (g L − 1 ) at the beginning and after x days, respectively. C 1 and C x were the lipid contents (%) following the first measurement and after x days, accordingly. It represents the whole duration of incubation. 2.3 Protein, carbohydrate, chlorophyll and carotenoid quantification Protein: BCA assay was performed on centrifuged pellets (5 mL culture, 4000 × g , 5 minutes) that were rinsed three times with deionized water before use. The absorbance was quantified at 562 nm (Lin et al. 2024 ). Carbohydrates: The phenol-sulfuric acid technique was employed. Samples underwent centrifugation (4000 × g , 5 minutes), were washed, and subjected to a subsequent centrifugation process three times. The absorbance was quantified at 490 nm (Chen et al. 2023 ). Photosynthetic pigments: Pellets of cells from 5 mL of culture were extracted using 95% acetone at 4°C for 12 h in darkness. Absorbance at 470, 646, and 663 nm was used to calculate concentrations (Roldan-Prieto et al. 2024 ). 2.4 Analysis of photosynthetic performance Photosynthetic characteristics were assessed utilizing a PEA fluorometer (Hansatech, UK). Dark-adapted samples (2 mL, 15 min) and the highest photochemical productivity Fv/Fm of algal cells SPII, the quantity of light received per activity response center (ABS/RC), the excitation energy harnessed each activity reaction concentrate (TRo/RC), an electron transportation rate (ETo/RC), the terminal acceptor for reducing the electrons flow at the PSI receptors (REo/RC), the effective movement of electrons from intersystem electron carriers to the electron acceptors on the intersystem PSI receptor side (REo/ETo), the quantified output of electron travel (φEo ETo/ABS), the decrease of PSI terminal recipients, the exciton energy conservation (PI total), the level of effectiveness and the likelihood of electrons transport (ψo ETo/TPo), and other associated photographers variables were computed utilizing the PEA Plus software (Hansatech) (Cheng et al. 2024 ; Zhang et al. 2016 ). 2.5 Metabolomics analysis Cells from CK, 0.4 g L − 1 glycerol, and 0.5 g L − 1 glucose treatments (day 10) were rapidly refrigerated in nitrogen gas and preserved at − 80°C. Biotree Biotech (Shanghai) conducted non-targeted metabolomics. Nonpolar metabolites were detected utilizing a Vanish Thermo Fisher Scientific Corporation system and the Kinetex C-18 Liquid Column was used to separate the compounds of interest. Liquid chromatography phase A is t water phase with 0.01% acetone as the solvent, whereas phase B consists of a 1:1 (v/v) mixture of isopropyl alcohol and acetonitrile. The sample tray temp was established at 4°C, and volume of injection was 2 µL. Mass spectra analysis was conducted utilizing the Orbitrap Exploris 120 System, employing Xcalibur (version 4.4; Thermo Fisher Science) control program for the gathering of primary and secondary mass spectrometry data. 2.6 Statistical analysis Data represent mean ± SD of ≥ 3 replicates in biology. Tukey's post-hoc test in conjunction with ANOVA (IBM SPSS 24) identified significant differences ( p < 0.05). Graphs were generated using OriginPro 2024. 3 Results 3.1 Effects of carbon sources on biomass and yield of Tribonema minus. Glycerol supplementation significantly enhanced Tribonema minus growth compared to the control (CK), with biomass increasing proportionally to cultivation time (Fig. 1 A). The 0.4 g L − 1 glycerol treatment yielded peak biomass (1.81 g L − 1 ), significantly surpassing other concentrations ( p < 0.001, F = 28.6). Biomass decreased dramatically at 1.0 g L − 1 glycerol (Fig. 1 A), indicating 0.4 g L − 1 as the optimal concentration. After 10 days, biomass productivity reached 0.11 g L − 1 d − 1 at 0.4 g L − 1 glycerol — 2.7-fold higher than CK (Fig. 1 B). Both suboptimal (0.2 g L − 1 ) and supraoptimal (1.0 g L − 1 ) concentrations reduced productivity. Complementing these findings, glucose exhibited superior efficacy. The 0.5 g L − 1 glucose treatment achieved maximal biomass (2.60 g L − 1 ), representing a 3.06-fold increase vs. CK (0.85 g L − 1 ) (Fig. 1 C). Biomass productivity peaked at 0.18 g L − 1 d − 1 under 0.5 g L − 1 glucose (Fig. 1 D). Analogous to glycerol, supraphysiological glucose concentrations (≥ 0.60 g L − 1 ) inhibited growth. 3.2 Effects of carbon sources on lipids, proteins, and carbohydrates Under heterotrophic conditions, glycerol supplementation significantly enhanced lipid synthesis in T. minus (Figs. 2 A, 2 B). The 0.4 g L − 1 glycerol treatment yielded maximal lipid content (48.1%) and productivity (0.056 ± 0.002 g L − 1 d − 1 ) after 10 days, surpassing other concentrations ( p < 0.05) (Figs. 2 A, 2 B). Supraoptimal glycerol levels (≥ 0.6 g L − 1 ) reduced lipid content to 39.4% and productivity to 0.022 ± 0.001 g L − 1 d − 1 . In glucose-treated cultures, lipid content increased concentration-dependently, peaking at 50.9% (0.5 g L − 1 ) (Fig. 3 A). Lipid productivity peaked at 0.089 ± 0.002 g L − 1 d − 1 (0.5 g L − 1 ), though growth rates declined at higher concentrations (Fig. 3 B). Protein synthesis responded differentially to carbon sources. The 0.4 g L − 1 glycerol group achieved maximal protein content (900.91 mg L − 1 ), significantly exceeding the control (181.61 mg L − 1 ) (Fig. 2 C). For glucose, 0.5 g L − 1 and 0.6 g L − 1 treatments yielded 725.16 mg L − 1 and 695.34 mg L − 1 (Fig. 3 C), with stable levels across concentrations. Carbohydrate accumulation patterns diverged markedly. Glycerol treatments peaked at 0.8 g L − 1 (392.13 mg L − 1 ) (Fig. 2 D). Glucose induced concentration-proportional increases (Fig. 3 D), with 0.5 g L − 1 yielding 2.53-fold higher carbohydrates than control. 3.3 Carbon source regulation of photosynthetic efficiency The photosynthetic response of T. minus to glycerol and glucose was assessed by Fv/Fm ratios and OJIP luorescence of chloroplasts transients (Xu et al. 2022 ). Fv/Fm values in control (CK) and glycerol-treated groups remained at 0.6–0.8 throughout cultivation (Fig. 4 A), whereas glucose-treated groups showed stable values (Fig. 5 A). Conversely, supraoptimal glycerol (≥ 0.8 g L − 1 ) and suboptimal glucose (0.2 g L − 1 ) reduced Fv/Fm by 18–22% ( p < 0.01), signifying carbon source-induced photoinhibition. Transients of fluorescent chlorophyll were employed to examine how two carbon sources affected T. minus's photosynthetic activity. Figure 4 B illustrates that the fluorescence intensity at each phase of the OJIP curve (J, I, P) exhibited significant changes with the prolongation of incubation time following glycerol addition, signifying glycerol supplementation accelerated electron transfer from QA to PSI, evidenced by J-I phase contraction (Fig. 4 B). The standardized OJIP flash curve (Fig. 4 C) demonstrates the decrease during he J-I period, signifying an enhancement in electron transfer efficiency from QA to PSI. The OJIP curve for the glucose treatment group exhibited a larger value at the P point (highest fluorescence intensity) (Fig. 5 B,C), signifying an enhancement in electron acceptor capacity. In the subsequent phase of incorporating suitable concentrations of glycerol (Fig. 4 D) and glucose (Fig. 5 D), both excited storage of energy (TRo/RC) and sensitivity to light (ABS/RC) exhibited an increase, signifying that the external organic carbon source supplied supplementary reducing power and energy to the cell, thereby enhancing the efficiency of multiple stages within the electron transport chain. However, late-stage declines in electron transfer efficiency (ETo/RC) reflected nutrient depletion-induced metabolic stress, consistent with density-dependent PSII inhibition in microalgae. (Goncalves et al. 2016 ). 3.4 Carbon source effects on photosynthetic pigments Glycerol supplementation significantly altered photosynthetic pigment profiles in Tribonema minus (Fig. 6 A). Chlorophyll a content increased across all glycerol-treated groups compared to the control group (CK, 6.13 mg L − 1 ), with 0.4 g L − 1 glycerol yielding peak enhancement (8.20 mg L − 1 , p < 0.05). In contrast, chlorophyll b exhibited minimal response to glycerol treatments (Fig. 6 A), maintaining consistent levels. Carotenoid content peaked at 0.2–0.4 g L − 1 glycerol (2.62 ± 0.15 mg L − 1 ). Compared to glycerol, glucose supplementation exerted a more pronounced effect on chlorophyll a content in Tribonema minus (Fig. 6 B). Chlorophyll a concentrations across all glucose-treated groups were markedly reduced compared to the control group (CK, 6.13 mg L − 1 ), with the minimum value observed at 0.3 g L − 1 glucose (4.28 mg L − 1 ) ( p < 0.01), followed by 0.4 g L − 1 glucose (4.94 mg L − 1 ). Conversely, chlorophyll b synthesis responded positively to glucose supplementation. After 10 days, the 0.6 g L − 1 glucose treatment yielded significantly higher chlorophyll b (6.80 mg L − 1 ) than CK (5.93 mg L − 1 ) ( p < 0.05). Concentrations of 0.4–0.5 g L − 1 glucose also enhanced chlorophyll b production (6.78–6.79 mg L − 1 ). Carotenoid content remained stable across glucose concentrations (1.39–1.67 mg L − 1 ), indicating preserved photoprotective function regardless of carbon availability. 3.5 Extraction of metabolites and study of pathways at optimal concentration of carbon sources To clarify the metabolic mechanisms esponsible for glycerol- and glucose-induced growth and lipid increase in T. minus , we conducted non-targeted metabolomic analysis at optimal carbon source concentrations (0.4 g L − 1 glycerol and 0.5 g L − 1 glucose). Principal component analysis (PCA) revealed significant metabolic divergence between treatment groups and the control (CK), with clear separation in the multivariate space (Fig. 7 A). The glycerol-treated group (GY) showed 64.4% and 29.9% variance explained by PC1 and PC2, respectively, indicating profound metabolic reprogramming induced by glycerol supplementation. Similarly, the glucose-treated group (PTT) exhibited substantial metabolic differentiation from CK (37.1% and 14.7% variance for PC1 and PC2), though with lower cumulative variance. Additionally, the differential metabolites were quantified and analyzed using volcano plots. Figure 7 C illustrates the differential metabolites between the CK and GY groups, identifying a total of 477 metabolites, comprising 125 that were up-regulated and 352 that were down-regulated. This includes terpenoids (195), shikimic acid and phenylpropanoid pathway compounds (99), fatty acids (149), alkaloids (142), amino acids and peptides (54), carbohydrates (35), and polyketides (35). While the CK and PTT groups exhibited 64 up-regulated and 207 down-regulated metabolites (Fig. 7 D), including terpenes (70), shikimic acid and phenylpropionic acid (56), fatty acids (30), carbohydrates (23), amino acids and short peptides (14). To further determine the differential regulation of metabolic pathways by glycerol and glucose treatments in T. minus , differential metabolites were screened (Table S1 ), which showed that the differential metabolites in the GY and PTT groups versus the CK group were mainly enriched in glycolysis/glycolysis, fatty acid metabolism, TCA cycle, pyruvate metabolism, and terpene skeleton biosynthesis (Fig. 8 ). Glycerol treatment significantly promoted the metabolic up-regulation of metabolites in the second half of glycolysis, such as glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), (Log 2 FC = 5.38 and 3.70, respectively). In contrast, glucose treatment primarily enriches metabolites in the early glycolysis phase, such as the increase of citric acid as well as fusoic acids, sulfuric acid in the TCA cycle also promoted the activation of fatty acid synthesis pathways, resulting in a significant increase in important polyunsaturated fatty acids such as including docosahexaenoic acid, eicosatetraenoic acid, linoleic acid, α-linolenic acid, and eicosapentaenoic acid. 4 Discussion This study shows that an appropriate supplementation of carbon sources can significantly enhance the proliferation of T. minus , but excessively high levels of carbon sources lead to metabolic burdens and inhibitory effects, a phenomenon further confirmed by metabolomics results. 4.1 Regulatory Mechanisms of Carbon Sources on the Growth and Component Synthesis of Tribonema minus The promoting effects of glycerol and glucose on biomass accumulation confirm that exogenous organic carbon sources can effectively compensate for the insufficient photosynthetic carbon fixation under heterotrophic conditions. However, the concentration thresholds at which the two reach their maximum biomass are different (glycerol: 0.4 g L − 1 ; glucose: 0.5 g L − 1 ), and the peak biomass of glucose is higher. This is mainly attributed to the differences in their metabolic pathways: as a monosaccharide, glucose can be rapidly absorbed by cells through glucose transporters and directly enter the glycolysis pathway, efficiently providing energy and the precursors needed for anabolism (Chen et al. 2020 ). In contrast, glycerol must first be converted into pyruvate through a phosphorylation pathway, and this additional step may limit its rate of energy supply. Metabolomics data support this view, showing that glucose treatment significantly increases the levels of intermediates in glycolysis and the TCA cycle, reflecting an overall enhancement of central carbon metabolism flux. It is noteworthy that excessively high concentrations of any carbon source can inhibit the growth of T. minus , but the underlying mechanisms may differ. Excess glycerol induces osmotic stress, disrupting energy metabolism and growth homeostasis (Liu et al. 2021 ); analogous to glycerol, supraphysiological glucose concentrations (≥ 0.6 g L − 1 ) inhibited growth, this is due to the excessive input of glucose, which can lead to the abnormal accumulation of intermediate metabolites in glycolysis and the tricarboxylic acid cycle, causing an imbalance in intracellular carbon flux and the accumulation of metabolic by-products (such as pyruvate and lactate), thereby reducing carbon utilization efficiency and potentially inducing metabolic toxicity (Zhou et al. 2021 ). In summary, an appropriate amount of carbon sources can significantly enhance the proliferation of T. minus , but excessively high levels of carbon sources can cause metabolic burdens and inhibitory effects, a phenomenon further validated by subsequent metabolomics results. In terms of component synthesis, this “low promotion and high inhibition” phenomenon is particularly evident in lipid synthesis. The optimal concentrations of glycerol and glucose can maximize lipid yield because they are effective sources of acetyl-CoA, a key precursor for lipid synthesis. While excess disrupts NADPH/ATP balance, inhibiting fatty acid synthase (Zhang et al. 2025 ; Pasciu et al. 2021 ). Although glucose efficiently drives lipid synthesis, supraoptimal levels induce metabolic burden from byproduct accumulation under oxygen-limited conditions (Huang et al. 2021 ), confirming carbon source optimization critically regulates microalgal lipid output (Kakarla et al. 2018 ; Ning et al. 2020). In terms of protein and carbohydrate synthesis, carbon sources also exhibit different directing effects. The significant increase in protein content under glycerol treatment, this reflects glycerol-3-phosphate serving as serine precursor for amino acid synthesis (Igamberdiev et al. 2018). And the stability of protein levels under glucose treatment, this stability suggests glucose-derived α-ketoglutarate sustains TCA cycle flux, maintaining protein homeostasis (Li et al. 2022 ) and potential mTOR pathway prioritization (Zhang et al. 2025 ). The differences in carbohydrate accumulation patterns are more pronounced: under high glycerol concentrations, G3P may be more diverted to lipid biosynthesis, limiting carbohydrate accumulation (Ma et al. 2022 ); whereas glucose can be directly converted into glucose-6-phosphate (G6P) for efficient storage carbohydrate synthesis, bypassing the TCA cycle required when using acetate as a carbon source (Joun et al. 2023 ). 4.2 The effect of carbon sources on photosynthetic physiology Under heterotrophic conditions, the addition of exogenous carbon sources has complex and interesting effects on the photosynthetic system. Overall, a suitable concentration of carbon sources maintains the maximum photochemical efficiency of PSII (Fv/Fm), indicating that its core photochemical reactions are not destroyed and cell viability is preserved (Li et al. 2025 ). However, stressful conditions (excessive glycerol or insufficient glucose) can trigger a significant decline in Fv/Fm, similar to the response of Phaeodactylum tricornutum under high salinity stress, where the integrity of chloroplast structure is protected by enhancing non-photochemical quenching (NPQ) and initiating chlorophyll degradation pathways, at the cost of reduced light absorption and increased thermal dissipation (Rautenberger et al. 2024; Uzlasir et al. 2023 ; Wang et al. 2019 ). This is an adaptive strategy to protect the photosynthetic apparatus under stress. Chlorophyll fluorescence transient (OJIP) analysis further reveals the differential regulation of the photosynthetic electron transport chain by carbon sources. Glycerol treatment accelerates the flow of electrons from QA to PSI (reflected by the contraction of the J-I phase), which may be because upon entering the cell, glycerol can be transformed into pyruvate via the phosphorylation pathway, which contributes to cellular respiration, increases the availability of reducing equivalents, and stimulates the synthesis of NADPH and ATP, thereby augmenting the efficiency of each phase of the electron transport chain and enhancing the accumulation of photosynthetic reducing power (Villanova et al. 2017 ). In contrast, glucose treatment showed an enhanced PSI end electron acceptor capacity (increased P point fluorescence intensity). This is because the ATP and NADH produced by glycolysis provide sufficient terminal electron acceptors for the photosynthetic electron transport chain, preventing the accumulation of electrons in the chain and thereby avoiding excessive production of reactive oxygen species (ROS) (Nikkanen et al. 2021 ). The findings indicate that the carbon supply influences electron transport efficiency and may modify the entire photosynthetic process of T. minus . 4.3 Regulatory Effect of Carbon Sources on Pigments of Tribonema minus The response of photosynthetic pigments to carbon sources further confirms the synergistic regulation of metabolism and light energy capture in cells. Glycerol promotes the synthesis of chlorophyll a, most likely because its derivative, 3-phosphoglyceric acid (3-PGA), provides the carbon skeleton for the synthesis of δ-aminolevulinic acid (ALA), a key precursor of chlorophyll a (Ma et al. 2022 ). Glucose, on the contrary, reduces the content of chlorophyll a. This is likely an active regulatory strategy, which reduces the amount of main photosystem pigments to lower the potential light excitation energy under heterotrophic metabolic states, thereby preventing the massive generation of ROS from the source to avoid oxidative damage (Gao et al. 2024 ). Meanwhile, glucose promoted the synthesis of chlorophyll b. This suggests that glucose availability optimizes light-harvesting complex development through enhanced metabolic activity (Ma et al. 2022 ; Liu et al. 2009 ). Under glucose limitation (≤ 0.3 g L − 1 ), pigment synthesis was significantly inhibited due to carbon skeleton scarcity, particularly affecting chlorophyll b which requires substantial energy investment (Zuliani et al. 2024 ). The stability of carotenoid content under different carbon source treatments highlights its indispensable antioxidant role in maintaining photosystem function and stress resistance (Zheng et al. 2020 ; Kato et al. 2017 ). 4.4 Analysis of carbon source-specific metabolic pathways Non-targeted metabolomics analysis reveals the deep metabolic mechanisms by which glycerol and glucose induce differential phenotypes from a systemic level. PCA shows that both carbon sources significantly alter cellular metabolic states through specific regulatory mechanisms. Glycerol, as a non-fermentable carbon source, enhances lipid biosynthesis and reducing power supply while promoting secondary metabolite accumulation (Pasciu et al. 2021 ; Zhang et al. 2025 ). In contrast, glucose primarily fuels glycolysis and TCA cycle activity, providing energy and precursor molecules through fundamentally different regulatory pathways (Patel et al. 2018 ). Pathway enrichment analysis revealed that the differential metabolites were mainly concentrated in central carbon metabolism and lipid synthesis pathways. Specifically, glycerol treatment significantly upregulated the levels of metabolites in the latter part of glycolysis. G3P is not only a key precursor for glycerophospholipid synthesis, but its accumulation also suggests that it may play a crucial role in regulating lipid biosynthesis (Xue et al. 2017 ). At the same time, the increase in TCA cycle intermediates (such as citrate and fumarate) provides ample carbon skeletons and reducing power for fatty acid synthesis, ultimately leading to the significant accumulation of various high-value polyunsaturated fatty acids. Therefore, glycerol and glucose as carbon sources significantly promote the lipid metabolism of T. minus , improve its lipid synthesis ability, and propose innovative concepts for utilizing microalgae in the generation of biofuels and high-value compounds (Zhao et al. 2021 ). 5 Summary In summary, adding appropriate concentrations of glycerol (0.4 gL − 1 ) and glucose (0.5 gL − 1 ) can effectively promote the proliferation and lipids buildup of T. minus , with glucose particularly outstanding in enhancing algal cell density and lipid yield. Further mechanistic exploration reveals that these two exogenous carbon sources not only significantly enhance the synthesis levels of proteins and carbohydrates, but also indirectly maintain the stability of the photosynthetic system by participating in photorespiration metabolism and energy supply. Non-targeted metabolomics results further indicate that carbon source treatment can upregulate key fatty acid synthesis pathways, thereby promoting lipid synthesis metabolism. In addition, we further studied the effects of glycerol and glucose on the particle size and potential of exosomes from T. minus . The results showed that the Zeta potentials of the exosomes in the glucose group (− 18.85 mV and − 16.69 mV) were both higher than that of the control group (− 20.75 mV), indicating a decrease in the absolute value of the surface charge of the exosomes. This suggests that the addition of carbon sources may affect the distribution of charged groups on the exosomal membrane surface. In terms of particle size, the average particle size of exosomes in the glucose group was 204.2 nm, which was smaller than the control group (224.1 nm), while the glycerol group significantly decreased to 125.0 nm, showing greater uniformity (PI = 0.19). The above results indicate that exogenous carbon sources, particularly glycerol, can regulate the biophysical characteristics of exosomes from T. minus , optimizing their size distribution and surface charge, thereby providing an important material basis and regulatory basis for the application of microalgal-derived exosomes in drug delivery or functional formulations. Overall, glycerol and glucose significantly enhance the lipid production capacity of T. minus through the synergistic regulation of carbon flow distribution and energy metabolism, providing a viable nutritional regulation strategy for the utilization of microalgae as energy sources, with important application prospects in microalgae biofuel development. This study systematically explains the potential of external carbon sources in the high-value utilization of algae from the perspectives of metabolic regulation and exosome characteristics, laying a theoretical foundation for the practical application of algal biotechnology. Future research could further focus on the interaction mechanisms between carbon source concentration, cultivation conditions, and metabolic pathways, optimizing carbon source utilization efficiency through metabolic engineering techniques, thereby enhancing the application prospects of T. minus in energy and high-value compound synthesis. Declarations Acknowledgments The authors would like to acknowledge the Ningbo Team Science and Technology Special Correspondent Project (2024S217), the National Natural Science Foundation of China (32400299) and the Ningbo Natural Science Foundation (2024J166). Author’s contributions L.Y.C.: Writing-original draft, Conceptualization, and Writing-review & editing. 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Supplementary Files Supplementaryfile1.docx floatimage1.png Graphical Abstract Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 28 Dec, 2025 Reviews received at journal 28 Dec, 2025 Reviews received at journal 02 Dec, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers agreed at journal 09 Nov, 2025 Reviewers agreed at journal 08 Nov, 2025 Reviewers invited by journal 29 Oct, 2025 Submission checks completed at journal 27 Oct, 2025 First submitted to journal 18 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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02:37:19","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136245,"visible":true,"origin":"","legend":"","description":"","filename":"6837dc10be944d86b3a94959cb43c75e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/17fd8765795063ce92b18866.xml"},{"id":95692684,"identity":"4af376dd-31c8-47cd-bbb9-8675b6cf07a0","added_by":"auto","created_at":"2025-11-12 02:37:19","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142787,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/468c1f147d98212d1c22c4e8.html"},{"id":95799164,"identity":"05011e71-9bad-4ebc-b32b-6e6cb42540b1","added_by":"auto","created_at":"2025-11-13 08:18:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1029719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of varying glycerol concentrations on biomass (A) and production (B) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTribonema minus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and the effects on various glucose concentrations on the biomass (C) and production. The ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.05 denotes a significant difference, and the error value is the mean of the SD ± (n = 3).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/634f1b0cf5b2425c20c00892.png"},{"id":95692667,"identity":"0afb75f7-45b5-4112-95b4-7a872acb2aa0","added_by":"auto","created_at":"2025-11-12 02:37:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1319167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of varying glycerol concentration on the following biochemical constituents of T. minus: (A) quantity of lipids, (B) lipids efficiency, (C) amount of protein, and (D) amount of carbohydrates. The ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt;0.05 denotes a significant difference, and the error value is the mean of the SD ± (n = 3).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/f1f229a5690cbb102f13cd72.png"},{"id":95692664,"identity":"b9d7fbda-19aa-4f15-8b10-89973c3adc86","added_by":"auto","created_at":"2025-11-12 02:37:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1503304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of varying glucose concentration on the following biochemical constituents of T. minus: (A) quantity of lipids, (B) lipids efficiency, (C) amount of protein, and (D) amount of carbohydrates. The ** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05 denotes a significant difference, and the error value is the mean of the SD ± (n = 3).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/69205b14c18880fb1069e077.png"},{"id":95692669,"identity":"18addc3d-6036-477f-a0df-a49e3e87a5b5","added_by":"auto","created_at":"2025-11-12 02:37:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":911341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotosynthetic characteristics of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. minus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under different glycerol concentrations. (A) Maximum quantum yield of PSII Fv/Fm, (B) initial OJIP curve, which is (C) adjusted OJIP curve, with (D) PSII-related chlorophyll fluorescence parameters.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/c83ed9e0d5186112739d2894.png"},{"id":95692673,"identity":"be5d1222-0abc-4e6e-8b97-6b2e8aa67f39","added_by":"auto","created_at":"2025-11-12 02:37:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1059213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotosynthetic characteristics of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. minus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder different glucose concentrations. (A) Maximum quantum yield of PSII Fv/Fm, (B) initial OJIP curve, which is (C) adjusted OJIP curve, with (D) PSII-related chlorophyll fluorescence parameters.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/34d040b88de322598ab2aceb.png"},{"id":95692687,"identity":"d3db6204-ed2d-4d4a-9a33-5f52a8b911d0","added_by":"auto","created_at":"2025-11-12 02:37:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":589159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different glycerol concentrations (A) and glucose concentrations (B) on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. minus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e pigments.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/04c5134a44b13135272caf81.png"},{"id":95692671,"identity":"6399d69f-0aaf-4ef5-8241-ad4300143e6e","added_by":"auto","created_at":"2025-11-12 02:37:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":280659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCA (A/B) and volcano (C/D) of 0.4 g L\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e−1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e glycerol and 0.5 g L\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e−1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e glucose supplemented with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. minus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/9835b29bd0b99f8b2f8341f5.png"},{"id":95800227,"identity":"398ca37b-bb97-49f8-adfc-0387b4e6071c","added_by":"auto","created_at":"2025-11-13 08:21:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":749726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of glycolysis/glycolytic metabolism , TCA cycle, lipid metabolism and terpene skeleton biosynthesis pathways in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. minus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with 0.4 g L\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e−1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e glycerol and 0.5 g L\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e−1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e glucose for the tenth day.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/490b0739819cf98533299e7b.png"},{"id":95804739,"identity":"149dcb5a-f269-4c73-bf3c-fd890d614851","added_by":"auto","created_at":"2025-11-13 08:39:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9092069,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/e78256dc-55ab-425f-91ff-301f468efb53.pdf"},{"id":95799976,"identity":"84acee1d-3d7c-4caa-9418-ae8b2a68b22c","added_by":"auto","created_at":"2025-11-13 08:21:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1183715,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/18e8afdf3c8c0174fef04c32.docx"},{"id":95692668,"identity":"c2443ab3-c2cc-436e-93be-2d30007f9f5d","added_by":"auto","created_at":"2025-11-12 02:37:18","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1134500,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7739724/v1/471ed21fbca66a3dd1d4ad7d.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Strategies for carbon regulation to enhance the efficient enrichment of energy storage materials in Tribonema minus","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMicroalgae, as highly efficient photosynthetic autotrophic organisms, demonstrate significant promise in the advancement of energy from renewable sources as well as high-value-added products. Compared with traditional terrestrial crops, microalgae grow rapidly, produce high biomass yields, avoid using agricultural land and can employ wastewater and waste gas for cultivation substrates. (Bumbac et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Under carbon source regulation, microalgae can significantly enhance the accumulation of storage substances such as lipids (up to over 50% of dry cell weight) and polysaccharides (20\u0026ndash;60%) (Song et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These products can be further converted into biodiesel and aviation fuel with higher cetane numbers and better combustion performance (Arora et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Neag et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), highlighting the synthetic potential of microalgae under carbon source guidance. Some microalgae (such as \u003cem\u003eTribonema minus\u003c/em\u003e) possess an active glyoxylic acid cycle pathway, enabling them to efficiently utilize organic carbon sources and demonstrating significant carbon metabolism plasticity. Notably, \u003cem\u003eTribonema minus\u003c/em\u003e (a filamentous microalga of the Chrysophyta phylum) has emerged as a promising candidate for bioenergy and high-value chemical production due to its unique photosynthetic physiology and high energy storage capacity. Physiological studies demonstrate that \u003cem\u003eT. minus\u003c/em\u003e achieves lipid contents of 45\u0026ndash;55% of dry cell weight under nitrogen stress, with \u0026gt;\u0026thinsp;80% of fatty acids comprising C16\u0026ndash;C18 chains \u0026mdash; an ideal profile for biodiesel (Feng et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Critically, certain strains simultaneously accumulate triacylglycerol (TAG) and other high-value products, enabling combined energy and functional compound production in a single cultivation process (Davis et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These attributes render \u003cem\u003eT. minus\u003c/em\u003e ideal for microalgal biorefining.\u003c/p\u003e\u003cp\u003eCarbon metabolism regulation in \u003cem\u003eT. minus\u003c/em\u003e reveals remarkable plasticity. Under autotrophic conditions, polysaccharides dominate accumulation (35\u0026ndash;40% of dry weight), whereas sodium acetate supplementation in mixotrophic cultures increases lipid content to \u0026gt;\u0026thinsp;50% without compromising growth rate (Dong et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This adaptability stems from a unique metabolic network: metabolomic analyses confirm a functional glyoxylate cycle that directly converts acetate into lipid precursors (Wang et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCarbon source regulation is a key factor controlling microalgal growth and metabolic flux during energy storage compound biosynthesis (Oliveira et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although microalgae primarily utilize CO₂ through photosynthesis for growth and energy storage under natural conditions (Xie et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), inorganic carbon sources are often constrained by environmental CO₂ concentration, light intensity, and nutrient availability, limiting efficient compound enrichment (Luo. 2020). Consequently, recent studies focus on organic carbon supplementation (e.g glucose, glycerol, acetate) to redirect metabolic networks toward lipid and polysaccharide synthesis (Gao et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These compounds provide direct carbon skeletons and energy while indirectly modulating carbon flux distribution, reducing equivalent generation, and signaling pathways to enhance storage efficiency (Sun et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For instance, glucose (an easily assimilated monosaccharide) significantly boosts biomass and lipid content in many microalgae, whereas glycerol uniquely promotes lipid synthesis and osmotic regulation (Yu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Carbon source type and concentration further influence trophic modes (autotrophy, heterotrophy, mixotrophy), photosynthetic activity, and cellular component allocation, ultimately determining biomass and metabolite yields (Mondal et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite this progress, carbon source regulation mechanisms in \u003cem\u003eT. minus\u003c/em\u003e remain poorly understood, particularly regarding systematic optimization of energy storage compound enrichment and metabolic pathway modulation by organic carbon sources. Advances in metabolomics and transcriptomics now enable elucidation of molecular mechanisms underlying metabolic network remodeling and functional molecule accumulation in \u003cem\u003eT. minus\u003c/em\u003e under carbon source regulation-a pivotal scientific challenge in this field (Long. 2017).\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Algal culture\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eTribonema minus\u003c/em\u003e is from the Ningbo Science and Technology Innovation Center of Zhejiang University and is currently stored in the algae seed room of the Food Branch. Algal cells were cultured in 1 L flasks with starting infection of 0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and irradiated at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 90\u0026thinsp;\u0026plusmn;\u0026thinsp;5 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e intensity LED lamp, shaking 3 times a day to ensure uniform distribution.\u003c/p\u003e\n \u003cp\u003eAlgal cells cultivated to the exponential phase were seeded in Bold base medium (BBM) supplemented by varying quantities of glycerol and glucose at (0.2, 0.4, 0.6, 0.8 and 1.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and glucose at (0.2, 0.3, 0.4, 0.5 and 0.6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with other nutrient components unchanged: NaNO₃ 0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, MgSO₄\u0026middot;7H₂O 0.08 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, NaCl 0.03 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, K₂HPO₄ 0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, KH₂PO₄ 0.18 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, CaCl₂\u0026middot;2H₂O 0.03 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, H₃BO₃ 0.01 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, EDTA-Na₂ 0.05 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, KOH 0.03 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, ZnSO₄\u0026middot;7H₂O 8.82 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Co(NO₃)₂\u0026middot;6H₂O 0.49 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, MnCl₂\u0026middot;4H₂O 1.44 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, CuSO₄\u0026middot;5H₂O 1.57 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Na₂MoO₄\u0026middot;2H₂O 1.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, FeSO₄\u0026middot;7H₂O 4.98 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eCultures were incubated under LED illumination (90 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with a 12 h:12 h light:dark photoperiod at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Continuous aeration was provided via compressed air, and cultures were shaken manually three times daily. After 14 days of cultivation, algal cells grown in carbon-free medium served as the control group (CK). Samples were collected bi-daily for the study of carbon dioxide, total lipids, proteins, carbohydrates, and photosynthetic pigments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Assessment of biomass and total lipid content\u003c/h2\u003e\n \u003cp\u003eBiomass concentration was determined gravimetrically. iberglass screens with pore sizes of 0.45 \u0026micro;m were dried at 105\u0026deg;C for 12 h, desiccated to constant weight (DW₁), and reweighed. A 10 mL algal suspension (V) was passed through filters, subjected to three washes with deionized water, dried at 105\u0026deg;C for 12 hours, and allowed to cool to a constant weight (DW₂).\u003c/p\u003e\n \u003cp\u003eBiomass concentration was calculated as:\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:\\text{DW}\\left(\\text{g}{\\text{L}}^{\\text{-1}}\\right)\\text{=}\\frac{\\text{(}{\\text{DW}}_{\\text{2}}\\text{-}{\\text{DW}}_{\\text{1}}\\text{)}}{\\text{V}}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eCalculate the biomass yield P (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) utilizing the equation provided below y.\u003c/p\u003e\n \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$$\\:\\text{P}\\text{=}\\frac{\\text{(}{\\text{X}}_{\\text{t}}\\text{-}{\\text{X}}_{\\text{0}}\\text{)}}{\\text{t}}\\text{}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eX\u003csub\u003e0\u003c/sub\u003e and X\u003csub\u003et\u003c/sub\u003e denote the amount of biomass (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) after the initial and t days, respectively; t denotes the total incubation duration. The total lipid content of \u003cem\u003eTribonema minus\u003c/em\u003e was determined by vanillin phosphate chromogenic method, and the filtered algae waste was rinsed twice with ionized water, then dried in a freeze dryer for 72 h, and the lyophilized algae powder was stored in a freeze \u0026minus;\u0026thinsp;80\u0026deg;C refrigerator. In order to facilitate lipid extraction, fifty milligrams of algae powder were dissolved in a designated volume of deionized water, Subsequently, 2 ml of sulfuric acid were introduced. The solution was subjected to heating in a water bath at 90\u0026deg;C for a duration of 20 minutes. then allowed to cool to room temperature. Subsequently, 3 mL of vanillin phosphate chromogenic reagent was incorporated, thoroughly mixed, and allowed to develop at room temperature for 20 minutes. After then, the amount of absorption was taken at 530 nm, and the lipid content was calculated in comparison to the standard curve (Gao et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pinger et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). The lipid content is quantified as a percentage of dry biomass.\u003c/p\u003e\n \u003cp\u003eStandard Curve: y\u0026thinsp;=\u0026thinsp;1.423X\u0026thinsp;+\u0026thinsp;0.025 R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998 (3)\u003c/p\u003e\n \u003cp\u003eLipid production P (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) is determined as follows:\u003c/p\u003e\n \u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e$$\\:{\\text{\u0026Rho;}}_{\\text{lipid}}\\text{=}\\text{}\\frac{{\\text{DW}}_{\\text{x}}\\text{\u0026times;}{\\text{C}}_{\\text{x}}\\text{-}{\\text{DW}}_{\\text{1}}\\text{\u0026times;}{\\text{C}}_{\\text{1}}}{\\text{t}}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere lipid productivity (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) is indicated; DW represents microalgae biomass (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); Indicates lipid content (%); DW\u003csub\u003e1\u003c/sub\u003e and DW\u003csub\u003ex\u003c/sub\u003e represent the biomass of microalgae (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at the beginning and after x days, respectively. C\u003csub\u003e1\u003c/sub\u003e and C\u003csub\u003ex\u003c/sub\u003e were the lipid contents (%) following the first measurement and after x days, accordingly. It represents the whole duration of incubation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Protein, carbohydrate, chlorophyll and carotenoid quantification\u003c/h2\u003e\n \u003cp\u003eProtein: BCA assay was performed on centrifuged pellets (5 mL culture, 4000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 5 minutes) that were rinsed three times with deionized water before use. The absorbance was quantified at 562 nm (Lin et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eCarbohydrates: The phenol-sulfuric acid technique was employed. Samples underwent centrifugation (4000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 5 minutes), were washed, and subjected to a subsequent centrifugation process three times. The absorbance was quantified at 490 nm (Chen et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003ePhotosynthetic pigments: Pellets of cells from 5 mL of culture were extracted using 95% acetone at 4\u0026deg;C for 12 h in darkness. Absorbance at 470, 646, and 663 nm was used to calculate concentrations (Roldan-Prieto et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Analysis of photosynthetic performance\u003c/h2\u003e\n \u003cp\u003ePhotosynthetic characteristics were assessed utilizing a PEA fluorometer (Hansatech, UK). Dark-adapted samples (2 mL, 15 min) and the highest photochemical productivity Fv/Fm of algal cells SPII, the quantity of light received per activity response center (ABS/RC), the excitation energy harnessed each activity reaction concentrate (TRo/RC), an electron transportation rate (ETo/RC), the terminal acceptor for reducing the electrons flow at the PSI receptors (REo/RC), the effective movement of electrons from intersystem electron carriers to the electron acceptors on the intersystem PSI receptor side (REo/ETo), the quantified output of electron travel (\u0026phi;Eo ETo/ABS), the decrease of PSI terminal recipients, the exciton energy conservation (PI total), the level of effectiveness and the likelihood of electrons transport (\u0026psi;o ETo/TPo), and other associated photographers variables were computed utilizing the PEA Plus software (Hansatech) (Cheng et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Metabolomics analysis\u003c/h2\u003e\n \u003cp\u003eCells from CK, 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol, and 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose treatments (day 10) were rapidly refrigerated in nitrogen gas and preserved at \u0026minus;\u0026thinsp;80\u0026deg;C. Biotree Biotech (Shanghai) conducted non-targeted metabolomics. Nonpolar metabolites were detected utilizing a Vanish Thermo Fisher Scientific Corporation system and the Kinetex C-18 Liquid Column was used to separate the compounds of interest. Liquid chromatography phase A is t water phase with 0.01% acetone as the solvent, whereas phase B consists of a 1:1 (v/v) mixture of isopropyl alcohol and acetonitrile. The sample tray temp was established at 4\u0026deg;C, and volume of injection was 2 \u0026micro;L. Mass spectra analysis was conducted utilizing the Orbitrap Exploris 120 System, employing Xcalibur (version 4.4; Thermo Fisher Science) control program for the gathering of primary and secondary mass spectrometry data.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eData represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of \u0026ge;\u0026thinsp;3 replicates in biology. Tukey\u0026apos;s post-hoc test in conjunction with ANOVA (IBM SPSS 24) identified significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Graphs were generated using OriginPro 2024.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Effects of carbon sources on biomass and yield of \u003cem\u003eTribonema minus.\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eGlycerol supplementation significantly enhanced \u003cem\u003eTribonema minus\u003c/em\u003e growth compared to the control (CK), with biomass increasing proportionally to cultivation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol treatment yielded peak biomass (1.81 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), significantly surpassing other concentrations (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F\u0026thinsp;=\u0026thinsp;28.6). Biomass decreased dramatically at 1.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as the optimal concentration. After 10 days, biomass productivity reached 0.11 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol \u0026mdash; 2.7-fold higher than CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Both suboptimal (0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and supraoptimal (1.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) concentrations reduced productivity. Complementing these findings, glucose exhibited superior efficacy. The 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose treatment achieved maximal biomass (2.60 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), representing a 3.06-fold increase vs. CK (0.85 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Biomass productivity peaked at 0.18 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Analogous to glycerol, supraphysiological glucose concentrations (\u0026ge;\u0026thinsp;0.60 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) inhibited growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effects of carbon sources on lipids, proteins, and carbohydrates\u003c/h2\u003e\u003cp\u003eUnder heterotrophic conditions, glycerol supplementation significantly enhanced lipid synthesis in \u003cem\u003eT. minus\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol treatment yielded maximal lipid content (48.1%) and productivity (0.056\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) after 10 days, surpassing other concentrations (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Supraoptimal glycerol levels (\u0026ge;\u0026thinsp;0.6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) reduced lipid content to 39.4% and productivity to 0.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In glucose-treated cultures, lipid content increased concentration-dependently, peaking at 50.9% (0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Lipid productivity peaked at 0.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), though growth rates declined at higher concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eProtein synthesis responded differentially to carbon sources. The 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol group achieved maximal protein content (900.91 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), significantly exceeding the control (181.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). For glucose, 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatments yielded 725.16 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 695.34 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with stable levels across concentrations. Carbohydrate accumulation patterns diverged markedly. Glycerol treatments peaked at 0.8 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (392.13 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Glucose induced concentration-proportional increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), with 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yielding 2.53-fold higher carbohydrates than control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Carbon source regulation of photosynthetic efficiency\u003c/h2\u003e\u003cp\u003eThe photosynthetic response of \u003cem\u003eT. minus\u003c/em\u003e to glycerol and glucose was assessed by Fv/Fm ratios and OJIP luorescence of chloroplasts transients (Xu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fv/Fm values in control (CK) and glycerol-treated groups remained at 0.6\u0026ndash;0.8 throughout cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), whereas glucose-treated groups showed stable values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Conversely, supraoptimal glycerol (\u0026ge;\u0026thinsp;0.8 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and suboptimal glucose (0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) reduced Fv/Fm by 18\u0026ndash;22% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), signifying carbon source-induced photoinhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTransients of fluorescent chlorophyll were employed to examine how two carbon sources affected \u003cem\u003eT. minus's\u003c/em\u003e photosynthetic activity. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB illustrates that the fluorescence intensity at each phase of the OJIP curve (J, I, P) exhibited significant changes with the prolongation of incubation time following glycerol addition, signifying glycerol supplementation accelerated electron transfer from QA to PSI, evidenced by J-I phase contraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The standardized OJIP flash curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) demonstrates the decrease during he J-I period, signifying an enhancement in electron transfer efficiency from QA to PSI. The OJIP curve for the glucose treatment group exhibited a larger value at the P point (highest fluorescence intensity) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C), signifying an enhancement in electron acceptor capacity.\u003c/p\u003e\u003cp\u003eIn the subsequent phase of incorporating suitable concentrations of glycerol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), both excited storage of energy (TRo/RC) and sensitivity to light (ABS/RC) exhibited an increase, signifying that the external organic carbon source supplied supplementary reducing power and energy to the cell, thereby enhancing the efficiency of multiple stages within the electron transport chain. However, late-stage declines in electron transfer efficiency (ETo/RC) reflected nutrient depletion-induced metabolic stress, consistent with density-dependent PSII inhibition in microalgae. (Goncalves et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Carbon source effects on photosynthetic pigments\u003c/h2\u003e\u003cp\u003eGlycerol supplementation significantly altered photosynthetic pigment profiles in Tribonema minus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Chlorophyll \u003cem\u003ea\u003c/em\u003e content increased across all glycerol-treated groups compared to the control group (CK, 6.13 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), with 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol yielding peak enhancement (8.20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, chlorophyll \u003cem\u003eb\u003c/em\u003e exhibited minimal response to glycerol treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), maintaining consistent levels. Carotenoid content peaked at 0.2\u0026ndash;0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol (2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eCompared to glycerol, glucose supplementation exerted a more pronounced effect on chlorophyll \u003cem\u003ea\u003c/em\u003e content in Tribonema minus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Chlorophyll \u003cem\u003ea\u003c/em\u003e concentrations across all glucose-treated groups were markedly reduced compared to the control group (CK, 6.13 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), with the minimum value observed at 0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose (4.28 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), followed by 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose (4.94 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Conversely, chlorophyll \u003cem\u003eb\u003c/em\u003e synthesis responded positively to glucose supplementation. After 10 days, the 0.6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose treatment yielded significantly higher chlorophyll \u003cem\u003eb\u003c/em\u003e (6.80 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than CK (5.93 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Concentrations of 0.4\u0026ndash;0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose also enhanced chlorophyll \u003cem\u003eb\u003c/em\u003e production (6.78\u0026ndash;6.79 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Carotenoid content remained stable across glucose concentrations (1.39\u0026ndash;1.67 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating preserved photoprotective function regardless of carbon availability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Extraction of metabolites and study of pathways at optimal concentration of carbon sources\u003c/h2\u003e\u003cp\u003eTo clarify the metabolic mechanisms esponsible for glycerol- and glucose-induced growth and lipid increase in \u003cem\u003eT. minus\u003c/em\u003e, we conducted non-targeted metabolomic analysis at optimal carbon source concentrations (0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol and 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose). Principal component analysis (PCA) revealed significant metabolic divergence between treatment groups and the control (CK), with clear separation in the multivariate space (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The glycerol-treated group (GY) showed 64.4% and 29.9% variance explained by PC1 and PC2, respectively, indicating profound metabolic reprogramming induced by glycerol supplementation. Similarly, the glucose-treated group (PTT) exhibited substantial metabolic differentiation from CK (37.1% and 14.7% variance for PC1 and PC2), though with lower cumulative variance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, the differential metabolites were quantified and analyzed using volcano plots. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC illustrates the differential metabolites between the CK and GY groups, identifying a total of 477 metabolites, comprising 125 that were up-regulated and 352 that were down-regulated. This includes terpenoids (195), shikimic acid and phenylpropanoid pathway compounds (99), fatty acids (149), alkaloids (142), amino acids and peptides (54), carbohydrates (35), and polyketides (35). While the CK and PTT groups exhibited 64 up-regulated and 207 down-regulated metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), including terpenes (70), shikimic acid and phenylpropionic acid (56), fatty acids (30), carbohydrates (23), amino acids and short peptides (14).\u003c/p\u003e\u003cp\u003eTo further determine the differential regulation of metabolic pathways by glycerol and glucose treatments in \u003cem\u003eT. minus\u003c/em\u003e, differential metabolites were screened (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which showed that the differential metabolites in the GY and PTT groups versus the CK group were mainly enriched in glycolysis/glycolysis, fatty acid metabolism, TCA cycle, pyruvate metabolism, and terpene skeleton biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Glycerol treatment significantly promoted the metabolic up-regulation of metabolites in the second half of glycolysis, such as glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), (Log\u003csub\u003e2\u003c/sub\u003e FC\u0026thinsp;=\u0026thinsp;5.38 and 3.70, respectively). In contrast, glucose treatment primarily enriches metabolites in the early glycolysis phase, such as the increase of citric acid as well as fusoic acids, sulfuric acid in the TCA cycle also promoted the activation of fatty acid synthesis pathways, resulting in a significant increase in important polyunsaturated fatty acids such as including docosahexaenoic acid, eicosatetraenoic acid, linoleic acid, α-linolenic acid, and eicosapentaenoic acid.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study shows that an appropriate supplementation of carbon sources can significantly enhance the proliferation of \u003cem\u003eT. minus\u003c/em\u003e, but excessively high levels of carbon sources lead to metabolic burdens and inhibitory effects, a phenomenon further confirmed by metabolomics results.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Regulatory Mechanisms of Carbon Sources on the Growth and Component Synthesis of \u003cem\u003eTribonema minus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe promoting effects of glycerol and glucose on biomass accumulation confirm that exogenous organic carbon sources can effectively compensate for the insufficient photosynthetic carbon fixation under heterotrophic conditions. However, the concentration thresholds at which the two reach their maximum biomass are different (glycerol: 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; glucose: 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the peak biomass of glucose is higher. This is mainly attributed to the differences in their metabolic pathways: as a monosaccharide, glucose can be rapidly absorbed by cells through glucose transporters and directly enter the glycolysis pathway, efficiently providing energy and the precursors needed for anabolism (Chen et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, glycerol must first be converted into pyruvate through a phosphorylation pathway, and this additional step may limit its rate of energy supply. Metabolomics data support this view, showing that glucose treatment significantly increases the levels of intermediates in glycolysis and the TCA cycle, reflecting an overall enhancement of central carbon metabolism flux.\u003c/p\u003e\u003cp\u003eIt is noteworthy that excessively high concentrations of any carbon source can inhibit the growth of \u003cem\u003eT. minus\u003c/em\u003e, but the underlying mechanisms may differ. Excess glycerol induces osmotic stress, disrupting energy metabolism and growth homeostasis (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); analogous to glycerol, supraphysiological glucose concentrations (\u0026ge;\u0026thinsp;0.6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) inhibited growth, this is due to the excessive input of glucose, which can lead to the abnormal accumulation of intermediate metabolites in glycolysis and the tricarboxylic acid cycle, causing an imbalance in intracellular carbon flux and the accumulation of metabolic by-products (such as pyruvate and lactate), thereby reducing carbon utilization efficiency and potentially inducing metabolic toxicity (Zhou et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In summary, an appropriate amount of carbon sources can significantly enhance the proliferation of \u003cem\u003eT. minus\u003c/em\u003e, but excessively high levels of carbon sources can cause metabolic burdens and inhibitory effects, a phenomenon further validated by subsequent metabolomics results.\u003c/p\u003e\u003cp\u003eIn terms of component synthesis, this \u0026ldquo;low promotion and high inhibition\u0026rdquo; phenomenon is particularly evident in lipid synthesis. The optimal concentrations of glycerol and glucose can maximize lipid yield because they are effective sources of acetyl-CoA, a key precursor for lipid synthesis. While excess disrupts NADPH/ATP balance, inhibiting fatty acid synthase (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Pasciu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although glucose efficiently drives lipid synthesis, supraoptimal levels induce metabolic burden from byproduct accumulation under oxygen-limited conditions (Huang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), confirming carbon source optimization critically regulates microalgal lipid output (Kakarla et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ning et al. 2020). In terms of protein and carbohydrate synthesis, carbon sources also exhibit different directing effects. The significant increase in protein content under glycerol treatment, this reflects glycerol-3-phosphate serving as serine precursor for amino acid synthesis (Igamberdiev et al. 2018). And the stability of protein levels under glucose treatment, this stability suggests glucose-derived α-ketoglutarate sustains TCA cycle flux, maintaining protein homeostasis (Li et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and potential mTOR pathway prioritization (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The differences in carbohydrate accumulation patterns are more pronounced: under high glycerol concentrations, G3P may be more diverted to lipid biosynthesis, limiting carbohydrate accumulation (Ma et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); whereas glucose can be directly converted into glucose-6-phosphate (G6P) for efficient storage carbohydrate synthesis, bypassing the TCA cycle required when using acetate as a carbon source (Joun et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.2 The effect of carbon sources on photosynthetic physiology\u003c/h2\u003e\u003cp\u003eUnder heterotrophic conditions, the addition of exogenous carbon sources has complex and interesting effects on the photosynthetic system. Overall, a suitable concentration of carbon sources maintains the maximum photochemical efficiency of PSII (Fv/Fm), indicating that its core photochemical reactions are not destroyed and cell viability is preserved (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, stressful conditions (excessive glycerol or insufficient glucose) can trigger a significant decline in Fv/Fm, similar to the response of \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e under high salinity stress, where the integrity of chloroplast structure is protected by enhancing non-photochemical quenching (NPQ) and initiating chlorophyll degradation pathways, at the cost of reduced light absorption and increased thermal dissipation (Rautenberger et al. 2024; Uzlasir et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This is an adaptive strategy to protect the photosynthetic apparatus under stress.\u003c/p\u003e\u003cp\u003eChlorophyll fluorescence transient (OJIP) analysis further reveals the differential regulation of the photosynthetic electron transport chain by carbon sources. Glycerol treatment accelerates the flow of electrons from QA to PSI (reflected by the contraction of the J-I phase), which may be because upon entering the cell, glycerol can be transformed into pyruvate via the phosphorylation pathway, which contributes to cellular respiration, increases the availability of reducing equivalents, and stimulates the synthesis of NADPH and ATP, thereby augmenting the efficiency of each phase of the electron transport chain and enhancing the accumulation of photosynthetic reducing power (Villanova et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, glucose treatment showed an enhanced PSI end electron acceptor capacity (increased P point fluorescence intensity). This is because the ATP and NADH produced by glycolysis provide sufficient terminal electron acceptors for the photosynthetic electron transport chain, preventing the accumulation of electrons in the chain and thereby avoiding excessive production of reactive oxygen species (ROS) (Nikkanen et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The findings indicate that the carbon supply influences electron transport efficiency and may modify the entire photosynthetic process of \u003cem\u003eT. minus\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Regulatory Effect of Carbon Sources on Pigments of \u003cem\u003eTribonema minus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe response of photosynthetic pigments to carbon sources further confirms the synergistic regulation of metabolism and light energy capture in cells. Glycerol promotes the synthesis of chlorophyll a, most likely because its derivative, 3-phosphoglyceric acid (3-PGA), provides the carbon skeleton for the synthesis of δ-aminolevulinic acid (ALA), a key precursor of chlorophyll a (Ma et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Glucose, on the contrary, reduces the content of chlorophyll a. This is likely an active regulatory strategy, which reduces the amount of main photosystem pigments to lower the potential light excitation energy under heterotrophic metabolic states, thereby preventing the massive generation of ROS from the source to avoid oxidative damage (Gao et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Meanwhile, glucose promoted the synthesis of chlorophyll b. This suggests that glucose availability optimizes light-harvesting complex development through enhanced metabolic activity (Ma et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Under glucose limitation (\u0026le;\u0026thinsp;0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), pigment synthesis was significantly inhibited due to carbon skeleton scarcity, particularly affecting chlorophyll \u003cem\u003eb\u003c/em\u003e which requires substantial energy investment (Zuliani et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The stability of carotenoid content under different carbon source treatments highlights its indispensable antioxidant role in maintaining photosystem function and stress resistance (Zheng et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kato et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Analysis of carbon source-specific metabolic pathways\u003c/h2\u003e\u003cp\u003eNon-targeted metabolomics analysis reveals the deep metabolic mechanisms by which glycerol and glucose induce differential phenotypes from a systemic level. PCA shows that both carbon sources significantly alter cellular metabolic states through specific regulatory mechanisms. Glycerol, as a non-fermentable carbon source, enhances lipid biosynthesis and reducing power supply while promoting secondary metabolite accumulation (Pasciu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, glucose primarily fuels glycolysis and TCA cycle activity, providing energy and precursor molecules through fundamentally different regulatory pathways (Patel et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePathway enrichment analysis revealed that the differential metabolites were mainly concentrated in central carbon metabolism and lipid synthesis pathways. Specifically, glycerol treatment significantly upregulated the levels of metabolites in the latter part of glycolysis. G3P is not only a key precursor for glycerophospholipid synthesis, but its accumulation also suggests that it may play a crucial role in regulating lipid biosynthesis (Xue et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At the same time, the increase in TCA cycle intermediates (such as citrate and fumarate) provides ample carbon skeletons and reducing power for fatty acid synthesis, ultimately leading to the significant accumulation of various high-value polyunsaturated fatty acids. Therefore, glycerol and glucose as carbon sources significantly promote the lipid metabolism of \u003cem\u003eT. minus\u003c/em\u003e, improve its lipid synthesis ability, and propose innovative concepts for utilizing microalgae in the generation of biofuels and high-value compounds (Zhao et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"5 Summary","content":"\u003cp\u003eIn summary, adding appropriate concentrations of glycerol (0.4 gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and glucose (0.5 gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) can effectively promote the proliferation and lipids buildup of \u003cem\u003eT. minus\u003c/em\u003e, with glucose particularly outstanding in enhancing algal cell density and lipid yield. Further mechanistic exploration reveals that these two exogenous carbon sources not only significantly enhance the synthesis levels of proteins and carbohydrates, but also indirectly maintain the stability of the photosynthetic system by participating in photorespiration metabolism and energy supply. Non-targeted metabolomics results further indicate that carbon source treatment can upregulate key fatty acid synthesis pathways, thereby promoting lipid synthesis metabolism. In addition, we further studied the effects of glycerol and glucose on the particle size and potential of exosomes from \u003cem\u003eT. minus\u003c/em\u003e. The results showed that the Zeta potentials of the exosomes in the glucose group (\u0026minus;\u0026thinsp;18.85 mV and \u0026minus;\u0026thinsp;16.69 mV) were both higher than that of the control group (\u0026minus;\u0026thinsp;20.75 mV), indicating a decrease in the absolute value of the surface charge of the exosomes. This suggests that the addition of carbon sources may affect the distribution of charged groups on the exosomal membrane surface. In terms of particle size, the average particle size of exosomes in the glucose group was 204.2 nm, which was smaller than the control group (224.1 nm), while the glycerol group significantly decreased to 125.0 nm, showing greater uniformity (PI\u0026thinsp;=\u0026thinsp;0.19). The above results indicate that exogenous carbon sources, particularly glycerol, can regulate the biophysical characteristics of exosomes from \u003cem\u003eT. minus\u003c/em\u003e, optimizing their size distribution and surface charge, thereby providing an important material basis and regulatory basis for the application of microalgal-derived exosomes in drug delivery or functional formulations.\u003c/p\u003e\u003cp\u003eOverall, glycerol and glucose significantly enhance the lipid production capacity of \u003cem\u003eT. minus\u003c/em\u003e through the synergistic regulation of carbon flow distribution and energy metabolism, providing a viable nutritional regulation strategy for the utilization of microalgae as energy sources, with important application prospects in microalgae biofuel development. This study systematically explains the potential of external carbon sources in the high-value utilization of algae from the perspectives of metabolic regulation and exosome characteristics, laying a theoretical foundation for the practical application of algal biotechnology. Future research could further focus on the interaction mechanisms between carbon source concentration, cultivation conditions, and metabolic pathways, optimizing carbon source utilization efficiency through metabolic engineering techniques, thereby enhancing the application prospects of \u003cem\u003eT. minus\u003c/em\u003e in energy and high-value compound synthesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe authors would like to acknowledge the Ningbo Team Science and Technology Special Correspondent Project (2024S217), the National Natural Science Foundation of China (32400299) and the Ningbo Natural Science Foundation (2024J166).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u0026nbsp;\u003c/strong\u003eL.Y.C.: Writing-original draft, Conceptualization, and Writing-review \u0026amp; editing. M.Z.: Writing-original draft, Conceptualization, and Writing-review \u0026amp; editing. Y.C.B.:Investigation and Formal analysis. R.X. and B.B.D: Conceptualization, Methodology, Supervision, Funding acquisition, Formal analysis, and Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u0026nbsp;\u003c/strong\u003eThis work was supported by the Ningbo Team Science and Technology Special Correspondent Project (2024S217), the National Natural Science Foundation of China (32400299) and the Ningbo Natural Science Foundation (2024J166).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest \u0026nbsp;\u003c/strong\u003eThe authors declare no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBumbac M, Nicolescu CM, Zaharescu T, Gurgu IV, Bumbac C, Manea EE, Dumitrescu C (2024) Biodegradation Study of Styrene\u0026ndash;Butadiene Composites with Incorporated \u003cem\u003eArthrospira platensis\u003c/em\u003e Biomass. 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Acta Microbiologica Sinica 61(7): 1799-1815\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carbon source regulation, Lipid accumulation, Non-targeted metabolomics, Tribonema minus","lastPublishedDoi":"10.21203/rs.3.rs-7739724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7739724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eTribonema minus\u003c/em\u003e efficiently accumulates energy storage substances such as lipids and polysaccharides, demonstrating potential for bioenergy and high-value-added product development. Carbon source regulation is a key strategy to promote energy storage compound enrichment. This study evaluated how different amounts of glycerol and glucose affected the carbon dioxide, lipid composition, photosynthesis rate, and metabolic pathways of \u003cem\u003eTribonema minus\u003c/em\u003e. The results showed that as glycerol and glucose concentrations increased, the biomass and lipid yield of each treatment group increased linearly, Among them, 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol treatment markedly raised the lipid content (48.1%) and biomass (1.81 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose treatment showed a more significant promoting effect, and increased biomass 3.06-fold and lipid content 1.27-fold ver. control. In addition, glycerol and glucose significantly increased the protein content of microalgae, with 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol yielding the highest protein content (900.91 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Although photosynthetic efficiency decreased in the late culture stage of each treatment group, the photochemical efficiency (Fv/Fm) remained above 0.6 at all times. Non-targeted metabolomics analysis showed that the addition of 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycerol and 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose significantly promoted fatty acid synthesis by enhancing glycolysis and providing sufficient glycerol-3-phosphate precursors, emphasizing the positive role of these carbon sources in lipid accumulation. The results indicate that glycerol and glucose serve as excellent carbon sources for \u003cem\u003eT. minus\u003c/em\u003e culture, facilitating a cost-efficient method to enhance biomass and lipid yields for biofuel production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Strategies for carbon regulation to enhance the efficient enrichment of energy storage materials in Tribonema minus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 02:37:13","doi":"10.21203/rs.3.rs-7739724/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-28T05:19:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-28T05:16:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T11:06:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13040349887446736041621889402945781187","date":"2025-11-10T15:17:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28912976712820948717670465628541147129","date":"2025-11-09T14:11:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137886096996403202639944187239526186521","date":"2025-11-09T04:57:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-29T09:33:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-27T10:10:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2025-10-19T01:16:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b218ec96-268e-48d2-a9d5-3e5ba6f796e2","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T10:10:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 02:37:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7739724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7739724","identity":"rs-7739724","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}