Sustainable valorization of grape pomace via cellulose extraction and cultivation of Tetradesmus obliquus: a biorefinery approach

Grape pomace is a promising agro-industrial residue with significant potential for sustainable biorefinery applications due to its high content of bioactive compounds and nutrients. This study evaluated its use as a substrate for Tetradesmus obliquus cultivation in photobioreactors and its effects on biomass growth and biochemical composition. Three photobioreactors were operated using Bold’s Basal Medium (BBM): one supplemented with grape pomace extract obtained by acid hydrolysis (E1), another with grape pomace extract obtained by microwave-assisted ethanol extraction (E2), and a control containing only BBM. Microalgal cultivation was monitored for 30 days by measuring cell density, growth kinetics, and pH. After cultivation, lipid profile, protein content, mineral accumulation, antioxidant activity, and cellulose crystallinity were analyzed. All treatments exhibited typical growth kinetics, with an exponential growth phase between days 5 and 10 and maximum cell density on day 26. E2 promoted the highest biomass growth, reaching 1.34 × 10 7 cells mL −1 and a specific growth rate of 0.083 day −1. In contrast, E1 improved biomass quality by increasing protein content, antioxidant activity, and cellulose crystallinity, while reducing the proportion of saturated fatty acids. Grape pomace extracts effectively modulated the growth and metabolism of Tetradesmus obliquus. While E2 favored biomass productivity, E1 enhanced the biochemical characteristics of the biomass, demonstrating the potential of grape pomace as a sustainable substrate for microalgal cultivation and supporting its application in integrated biorefinery processes.

The global wine industry generates substantial amounts of agro-industrial residues, with grape pomace representing approximately 20%–25% of the total processed grape mass, corresponding to 8.49 million tonnes per year worldwide (Abreu et al., 2025). The management of this by-product, composed mainly of skins, seeds, and stalks, represents an environmental challenge due to its high organic load and seasonal accumulation. However, increasing demand for sustainable and environmentally friendly processes has driven the search for innovative strategies to valorize such residues within the framework of the circular bioeconomy (Abreu et al., 2024).

Grape pomace is a rich source of bioactive compounds, particularly phenolic compounds, dietary fibers, and lignocellulosic materials. Several studies have demonstrated its high content of phenolics such as gallic acid, caffeic acid, and p-coumaric acid, which contribute to its antioxidant potential and industrial relevance (Meini et al., 2021). Additionally, grape pomace extracts present antioxidant (Deaconu et al., 2024), anti-obesity (Han et al., 2025) anti-diabetic (Lanzi et al., 2016) and anti-inflammatory activities (Deaconu et al., 2024).

Also, some of the polyphenols present in grape pomace, such as resveratrol and epigallocatechin gallate present anti-viral and immune-boosting properties, being proposed as candidates for preventing SARS-CoV-2 (Mehany et al., 2021). Recent studies have reported the utilization of polyphenols as prebiotics in functional foods (Dos Santos et al., 2017). Species of the genus Tetradesmus, particularly Tetradesmus obliquus, have shown promising characteristics for biotechnological applications due to their high growth rates, robustness, and ability to accumulate valuable metabolites such as proteins (reaching 30%–40% on a dry-mass basis), lipids, and antioxidants (da Silva et al., 2024). The biorefinery concept has emerged as a sustainable strategy for biomass utilization, enabling the integrated production of fuels, chemicals, and materials from renewable resources while supporting circular bioeconomy principles through improved resource efficiency and waste reduction (Meini et al., 2021; Trossolo et al., 2025).

Furthermore, Tetradesmus demonstrates remarkable efficiency in bioremediation processes, effectively removing nutrients such as nitrogen and phosphorus from effluents, thereby contributing to wastewater treatment and the mitigation of environmental pollution (Scoculi de Lira et al., 2025). The metabolic adaptability of this microalga, which allows for the adjustment of its composition in response to different cultivation conditions, underscores its potential for large-scale optimization (Alwaleed et al., 2025). Within this context, the use of waste-derived substrates, such as wine pomace, can significantly reduce production costs and improve process Sustainability (Manso et al., 2025). Furthermore, they can produce valuable compounds such as proteins, lipids, and pigments, making them suitable for integrated biorefinery processes (Trossolo et al., 2025).

Despite the growing interest in both grape pomace valorization and microalgal biotechnology, the integration of these approaches remains underexplored. Therefore, this study aims to investigate the sustainable valorization of grape pomace through its application as a substrate for the cultivation of T. obliquus, focusing on biomass growth rate and cellulose recovery within an integrated biorefinery approach.

Preparation of grape pomace flour (GPF)

The grape pomace (GP) samples from the ‘Touriga Nacional’ were supplied by the Carmim Cooperative Winery, located in the municipality of Reguengos de Monsaraz, in the Alentejo region of Portugal. Upon receipt, the samples were dried in an oven (J.P. Selecta, Barcelona, Spain) for 24 h at 60 °C, ground in a household blade grinder (Moulinex, Alençon, France) to obtain the flours, and finally stored in properly sealed polypropylene bags until use. The bags containing the GPF were transported to Brazil under appropriate conditions and stored away from light, heat, and humidity until the experimental growth medium were prepared (Piccoli et al., 2024).

Extracts preparation

The following conditions were used to prepare extract 1 (biomass E1): In a flask, 6 g of grape pomace was mixed with 18 mL of a 0.8% H 2 SO 4 (aq) solution. The mixture was heated to 90 °C for 1 h, cooled to room temperature, and then filtered under vacuum (Çakir et al., 2024). For the preparation of extract 2 (biomass E2), the optimal operating conditions were: microwave power (P), 408 W; solvent, 24% aqueous ethanol; solvent-to-solid ratio, 24/1 mL/g; extraction time, 5 min. Whereas 6 g of extract was added, the target concentration for using grape pomace was 0.2 g/L, assuming 30-liter reactors (Tsali and Goula, 2018). Extract E1 was added as supplement to BBM to photobioreactor 1 and the volume was adjusted to 30 L with BBM culture medium; the same procedure was followed with extract E2 in photobioreactor 2. No extract was added to photobioreactor 3, which was used as a control.

Cultivation of the microalgae Tetradesmus obliquus

The Tetradesmus obliquus strain CICLO_00125 (GenBank Accession Number PX631946) microalgae (Figure 1) was sourced from Startup Ciclo Biotecnologia Ambiental, Pelotas, RS, Brazil. Cultivation was carried out in 30 L photobioreactors using Basal Bold Medium (BBM) culture medium (Guillard and Lorenzen, 1972) with a stable pH range of 7–8. The culture was maintained at 22 °C ± 2 °C, with aeration provided by a Hailea (Zhejiang, China) 70 L/min electromagnetic aeration pump, with porous stones serving as air diffusers.

Illumination was achieved using 40 W fluorescent lamps, following a 16/8-h light/dark photoperiod to ensure optimal growth conditions and 100 µmol photons m -2 s -1. Microalgal cultivation was monitored for 30 days through cell density, growth kinetic and pH variation. The cultivation was carried out in parallel in different reactors, only one photobioreactor per experiment. The initial inoculum presented a cell density of (1.06 × 10 7). mL −1.

Biomass characterization analysis

Protein

The protein content was quantified using the Kjeldahl method (AOAC, 2016): 200 mg of microalgal biomass were digested with a catalytic mixture of 7:1 sodium sulfate and copper sulfate and 10 mL of concentrated sulfuric acid at 350 °C for 4 h in a digestion block. After digestion, 50 mL of distilled water and 40 mL of 50% sodium hydroxide were added for distillation. The released ammonia was captured in 30 mL of 4% boric acid containing a mixed indicator of methyl red and bromocresol green and, subsequently, titrated with standardized 0.1 mol L -1 hydrochloric acid. A blank was processed under the same conditions. Total protein content was calculated as nitrogen × 6.25 according to AOAC (2016).

Carbohydrates

Total carbohydrates were determined by the phenol-sulfuric acid method (Tan et al., 2024): 500 µL of aqueous solutions of dry biomass (1 mg mL -1) were reacted with 500 µL of 5% phenol and 2.5 mL of concentrated sulfuric acid. The reaction mixture was incubated for 5 min at 90 °C, cooled on ice for 5 min, and the absorbance was measured spectrophotometrically at 490 nm. Glucose was used as a calibration standard, and the results were expressed in milligrams of carbohydrate per Gram of dry biomass.

Pigments

Chlorophyll a and chlorophyll b were extracted and quantified according to the spectrophotometric method described by Jeffrey and Humphrey (1975): 25 mg of dry biomass was homogenized in 10 mL of 90% acetone, vortexed for 5 min, and centrifuged at 4,000 × g for 10 min. The supernatant was collected, and absorbance was measured at 647, 664, and 750 nm using a UV-Vis. Chlorophyll concentrations were calculated using the Jeffrey and Humphrey equations, with results expressed in mg g -1 of dry biomass (Jeffrey and Humphrey,1975).

Total carotenoids were determined following the method of Leitzke et al. (2025) and Rodríguez-Amaya (2001): 50 mg of dried biomass were extracted with 6 mL of chilled acetone and vortexed for 3 min. The mixture was transferred to a separatory funnel, followed by the addition of 2 mL of acetone, 3 mL of hexane, and 3 mL chilled distilled water. After phase separation, the polar phase was discarded, and the hexane phase was washed three times with 3 mL of distilled water. The final extract was transferred to a 5 mL amber volumetric flask and completed with hexane. Total carotenoids were then quantified spectrophotometrically at 450 nm, based on the extinction coefficient of β-carotene in hexane.

Ashes

The ash content was quantified gravimetrically following standard analytical protocols (AOAC, 2016); 1 g of dry biomass was weighed into previously incinerated porcelain crucibles and incinerated in a muffle furnace at 550 °C for 4 h. The resulting ash fraction was calculated as the percentage of inorganic residue relative to the initial mass of dry biomass.

Lipid analysis and fatty acid methyl esters profile

Lipid extraction

Total lipids were extracted using the Bligh and Dyer (1959) and quantified gravimetrically: 200 mg of dried biomass were mixed with 5 mL of chloroform, 10 mL of methanol, and 5 mL of 1.5% sodium sulfate solution and kept under agitation at room temperature for 1 h. Subsequently, 5 mL of chloroform and 5 mL of 1.5% sodium sulfate solution were added, followed by an additional 1 min of agitation. After phase separation, the biomass was re-extracted three times with 5 mL of a chloroform–methanol (1:2) solution and 1 min of agitation for each extraction. All nonpolar phases were pooled, and the solvent was evaporated to dryness. The resulting extract was redissolved in 2 mL of hexane and evaporated again. The dried extract was weighed and subjected to derivatization with boron trifluoride according to Dos Santos et al. (2021): the extract was refluxed for 5 min with 5 mL of 2% methanolic NaOH, followed by the addition of 5 mL of methanolic boron trifluoride solution and a second 3 min reflux. Then, 3 mL of saturated NaCl solution and 20 mL of hexane were added, and the nonpolar phase was separated, dried, and redissolved in HPLC-grade hexane.

Fatty acid methyl esters profile

The samples were analyzed using a gas chromatograph coupled with a mass spectrometer, model QP 2020 (Shimadzu, Kyoto, Japan), equipped with an Rtx-5MS capillary column (30 m × 0.25 mm × 0.25 μm) and helium as the carrier gas. The parameters were selected according to a methodology adapted from Berneira et al. (2021). The injection volume was 1 μL in split mode (1:50). Helium was used as the carrier gas at a constant flow rate of 1.20 mL min-1. The injector temperature was set to 200 °C, while the temperatures of the interface and the ion source were 200 °C. Electron ionization (EI) was used, with a mass scan range of 35–700 (m/z). The initial oven temperature was 100 °C; the temperature then rose at a rate of 3 °C/min until reaching the final oven temperature of 250 °C, which was maintained for 5 min, and the analysis time was 55 min.

Identification and quantification of fatty acids

Identification and quantification of fatty acids were performed by external calibration using authentic FAME standard solutions. Calibration curves were prepared from certified FAME standard mixtures (Sigma-Aldrich, São Paulo, SP, Brazil) diluted in hexane. A seven-point calibration curve was constructed at concentrations of 2.5, 4.0, 7.5, 11.0, 14.5, 18.0, and 21.5 mg/mL. Total ion chromatograms were acquired, and compound identification was achieved by comparison of experimental spectra with the NIST library 2.0. The fatty acids present in the samples but absent in the FAME standard were quantified using similar fatty acids as references, which constitute a semi-quantitative analysis. Supplementary Table S1 shows the correspondence between each fatty acid not present in the standard and its similar counterpart used for quantification in each sample.

Metals

For the acid decomposition, approximately 250 mg of sample was weighed directly into borosilicate tubes. Subsequently, 5.0 mL of HNO 3 was added, the reflux system was attached, and the tubes were placed in a digestion block at 150 °C for 2 h. After this period, 1.0 mL of H 2 O 2 was added, and the solution was returned to the block for an additional 30 min at 150 °C. At the end, the samples were removed from the block, allowed to cool, transferred to polypropylene flasks, and diluted to a final volume of 20 mL with distilled water.

The determination of the Cr, Cu, Fe, Mn, Ni, and Zn, a flame atomic absorption spectrometer (F AAS), model AAnalyst 200 (Perkin Elmer, Singapore), was used, equipped with a hollow cathode lamp for each analyte to be determined (Lumina, Perkin Elmer) and a deuterium arc lamp for background correction. For Na and K, the same spectrometer was used, but in flame atomic emission mode (F AES). In addition, acetylene (Linde, Barueri, SP, Brazil) was used as the fuel gas, and compressed air as the oxidant gas. The burner height used was 0.5 cm, and the acetylene flow rate was 2.5 L min −1 for all analytes. The calibration curve was prepared in an aqueous medium acidified with 2% (v/v) HNO 3.

Antioxidant capacity of methanolic extract

The antioxidant capacity of the samples was evaluated using DPPH and ABTS radical scavenging assays, as well as the ferric reducing antioxidant power (FRAP) assay, following adaptations of previously described methodologies. In all antioxidant assays performed (DPPH, ABTS, and FRAP), antioxidant activity was quantified by constructing calibration curves using Trolox standards at different concentrations, analyzed under the same experimental conditions as the samples. The results were expressed as Trolox-equivalent (TE) antioxidant capacity, that is, in mmol of Trolox equivalents per Gram of sample (mmol TE/g). For extraction of the compounds, a total of 3 mL of methanol was added to 300 mg of each biomass sample, followed by vigorous vortex mixing for 30 s. The samples were then subjected to sonication in a water bath at room temperature for 45 min and subsequently centrifuged at 7,500 × g for 10 min. The extraction procedure was repeated three times under the same conditions. The collected supernatants were combined, evaporated to dryness, and reconstituted in 3 mL of methanol (Haoujar et al., 2019).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay

Radical scavenging activity was determined using the stable radical DPPH. DPPH solution was prepared in methanol and adjusted to an absorbance of 1.10 ± 0.02 at 517 nm. For the assay, 3.9 mL of DPPH solution were mixed with 0.1 mL of the sample extract. The mixture was homogenized and incubated in the dark at room temperature for 30 min. Absorbance was then measured at 517 nm using a UV-Vis spectrophotometer, with methanol as the blank. The decrease in absorbance was used as an indicator of antioxidant activity. A calibration curve was constructed with the equation y = 0.0005x – 0.0649 and an R-squared of 0.9941.

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)

The ABTS• + radical cation was generated by reacting ABTS (7 mM) with potassium persulfate (140 mM), and the solution was kept in the dark for 16 h prior to use. The solution was then diluted with ethanol to obtain an absorbance of 0.70 ± 0.05 at 734 nm. For the assay, 3.0 mL of ABTS solution were mixed with 30 µL of the sample extract. After homogenization, the mixture was incubated in the dark for 6 min. Absorbance was measured at 734 nm using ethanol as the blank. The reduction in absorbance was considered proportional to the antioxidant capacity of the sample. A calibration curve was constructed with the equation y = 0.0003x + 0.0422 and an R-squared of 0.998.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was used to evaluate the reducing capacity of the samples. The working FRAP solution was prepared by mixing acetate buffer (0.3 M, pH 3.6), TPTZ solution (10 mM in 40 mM HCl), and ferric chloride solution (20 mM). For the assay, 90 µL of the sample extract were mixed with 270 µL of distilled water and 2.7 mL of FRAP reagent. The mixture was homogenized and incubated in a water bath at 37 °C for 30 min. Absorbance was measured at 595 nm, and the increase in absorbance was directly related to the reducing power of the sample. A calibration curve was constructed with the equation y = 0.0012x – 0.0376 and an R-squared of 0.997.

Determination of total phenolic content (TPC)

The TPC of the samples was determined using the Folin–Ciocalteu colorimetric method, based on electron transfer in alkaline medium from phenolic compounds to phosphomolybdic/phosphotungstic acid complexes, resulting in the formation of a blue chromophore measurable by spectrophotometry.

The reaction was performed as follows: an aliquot of 250 µL of sample extract or standard solution was transferred to a test tube. For the blank, 250 µL of methanol was used instead of the sample. Subsequently, 4000 µL of distilled water and 250 µL of Folin–Ciocalteu reagent (0.25 N) were added. The mixture was vortexed for 10 s and allowed to react for 3 min in the dark. Then, 500 µL of sodium carbonate solution (1 N) was added, followed by vortex mixing for an additional 10 s. The reaction mixture was incubated in the dark at room temperature for 2 h. After incubation, the absorbance was measured at 725 nm using a UV-Vis spectrophotometer.

Quantification of total phenolics was performed using a calibration curve constructed with gallic acid standard solutions at concentrations ranging from 0 to 200 μg/mL. The total phenolic content was expressed as milligrams of gallic acid equivalents per Gram of sample (mg GAE/g). All analyses were performed in triplicate, and the results were expressed as mean ± standard deviation.

Cellulose extraction

The process for cellulose extraction from microalgae biomass was adapted from Hamed et al. (2016). One Gram of microalgal biomass was weighed and transferred to a 500 mL beaker, followed by the addition of 250 mL of distilled water. The suspension was subjected to probe ultrasonication under the following conditions: 25 kHz frequency, 800 W power, for 30 min. The process was conducted in an ice bath to prevent overheating. After sonication, the suspension was centrifuged, and the recovered biomass was collected. The dried microalgal powder (1 g) was then treated with 25 mL of a toluene/ethanol mixture (68:32, v/v) under magnetic stirring for 24 h. Following filtration, the cell wall residue was treated with 25 mL of 24% (w/v) sodium hydroxide (NaOH) at 80 °C for 2 h to remove hemicelluloses. Subsequently, a bleaching step was performed using sodium hypochlorite solution (6%–10%) at pH 4.8, under agitation for 2 h at 70 °C. The resulting cellulose pellet was recovered by centrifugation, dried in an oven, and weighed.

After the extraction, the cellulose was characterized through Fourier transform infrared spectroscopy (FT-IR) was performed with potassium bromide in a Shimadzu spectrometer of infrared, model SPIRIT, with scanning from 4000 to 400 cm −1 with 45 scans and a resolution of 4 cm −1. Also, X-ray diffraction (XRD) patterns were recorded in a D8 ADVANCE, Bruker; equipped a copper tube (Cu), wavelength of 1.5418 Å; and operated at 40 kV and 40 mA. The analysis was performed in the range between 10 and 60 at scanning speed of 0.05 min −1. The crystallinity index (CrI) was calculated according to the Segal method (Segal et al., 1959), as expressed in Equation 1: where I200 is the maximum intensity of the crystalline diffraction peak corresponding to the (200) plane at 2θ = 22.5°, and Iam is the intensity of the amorphous region at 2θ = 18.5°. This method was used to estimate the relative proportion of crystalline and amorphous domains in the cellulose structure (Paniz et al., 2020).

Statistical analysis

All analyses regarding the characterization of the biomass obtained were conducted in triplicate, and the results are expressed as mean ± standard deviation. The chemical characterization and antioxidant activities were evaluated using one-way ANOVA followed by Tukey’s post hoc test (p < 0.05). All statistical analyses were performed using Statistica version 8.0 (Statistica Software, USA). The complete flowchart of the project development process is shown in Figure 2.

Tetradesmus obliquus culture and biomass production parameters

The cultivation parameters monitored over the 30-day period in the photobioreactors are presented in Figure 3. All experimental groups (control biomass, biomass E1: acid extract of grape pomace, and biomass E2: microwave-assisted ethanolic extract of grape pomace) exhibited a characteristic kinetic pattern: an initial lag phase during the first days, an exponential phase between days 5 and 10 (Figures 3A,B), followed by a progressive transition to the stationary phase, with peak cell density recorded on day 26 in all groups. The biomass E2 group showed the highest cell density values (1.34 × 10 7). mL -1 and µ = 0.083. day -1 from the fifth day of cultivation onward, followed by the control biomass group (1.11 × 10 7). mL -1 and µ = 0.072. day -1 and biomass E1 (9.15 × 10 6). mL -1 and µ = 0.05. day. -1

The higher biomass accumulation observed in the E2 group throughout the cultivation period suggests that the microwave-assisted ethanolic extract exerted a positive modulation on the growth of Tetradesmus obliquus without compromising photosynthetic activity. In contrast, extract E1, produced by acid hydrolysis with H 2 SO 4 at 90 °C, reduced cell density relative to the control throughout the cultivation period. In addition, the low concentration of hydrolysate used in this study (0.2 g L -1) likely did not sustain mixotrophic metabolism effectively. Regarding physicochemical parameters, pH increased progressively and homogeneously in all groups (Figure 3C), from 6.8 to values above 8.5 at the end of the experiment. No significant differences in pH dynamics appeared among the three treatments. Electrical conductivity (EC) showed minor variations throughout cultivation, without differentiation among groups (Figure 3D). The stabilization of cell density observed after day 26 occurred in all groups, including E2.

Physicochemical characterization

Figure 4A shows that biomass E1 presented chlorophyll a and b contents similar to those of the control biomass. In contrast, biomass E2 exhibited a significant reduction in both pigments. No significant differences (p > 0.05) were observed between the control and biomass E1 for chlorophyll a and b, whereas biomass E2 showed significantly lower values (p < 0.05). For β-carotene content (Figure 4B), no statistically significant differences (p > 0.05) were observed between the control biomass and biomass E1, nor between biomass E1 and biomass E2.

Regarding carbohydrate content (Figure 5A), no significant differences (p > 0.05) were observed among treatments E1, E2, and the control, with all groups maintaining values around 10%–11%. Protein analysis revealed that the control biomass and biomass E2 exhibited significantly lower protein contents compared to biomass E1. Biomass E1 showed the highest protein content among all microalgal samples (23.71% ± 0.57%). With respect to ash content, biomass E1 showed significantly lower values than the control (p < 0.05), while biomass E2 exhibited the lowest ash content among all microalgal biomasses, differing significantly from both the control and biomass E1.

Figure 6 shows the chromatograms of the fatty acids obtained from the control, E1, and E2 biomass samples. Supplementary Table S2 lists the identification of each peak. Supplementary Table S3 summarizes the quantification of the major fatty acids in the different microalgal biomasses, while Figure 5B shows the total percentages of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). Differences in total fatty acid concentration were observed among the treatments, with the control biomass presenting the highest content (22.42 mg/g of microalgae), followed by biomass E2 (18.49 mg/g) and biomass E1 (18.18 mg/g).

The control biomass exhibited a fatty acid profile dominated by polyunsaturated fatty acids. Methyl linolenate (C18:3) was the most abundant compound, followed by methyl linoleate (C18:2). In addition, methyl 4,7,10,13-hexadecatetraenoate (C16:4) was detected at considerable levels. Biomass E1 showed the lowest total fatty acid concentration (18.18 mg/g). Compared with the control, the relative contributions of the main PUFAs, C18:3 and C18:2, decreased, whereas the proportion of methyl 4,7,10,13-hexadecatetraenoate (C16:4) increased. Biomass E2 presented an intermediate total fatty acid concentration (18.49 mg/g). Its fatty acid profile was similar to that of the control biomass, with methyl linolenate (C18:3) and methyl palmitate (C16:0) remaining the predominant compounds.

The mineral composition of the samples is presented in Table 1. Overall, biomass cultivated with the acid extract (E1) showed the highest accumulation of minerals, particularly Cu, Na, and Zn. The control biomass showed the highest Fe concentration among the treatments, while biomass E1 was characterized by higher levels of Cu, Zn, and Na. Biomass E2 presented elevated concentrations of potassium (K) and manganese (Mn), indicating distinct mineral profiles according to the extraction strategy applied to grape pomace.

Antioxidant activities and total phenolic compounds

Figure 7 shows the total phenolic compounds (A) and antioxidant activities (B) of control biomass, E1, and E2. No significant differences (p > 0.05) were observed among the samples for total phenolic contents. For antioxidant activities (Figure 7B), different responses were observed depending on the method used. In the DPPH assay, E1 and E2 showed significantly higher values than the control (p < 0.05). In contrast, for ABTS, the control exhibited the highest activity, differing significantly from E1 and E2. Regarding FRAP, E2 showed the highest antioxidant activity, followed by the control, while E1 presented significantly lower values.

Cellulose extraction

The extraction of cellulose from microalgae cultivated in photobioreactors has been regarded as a promising strategy in the context of the circular bioeconomy due to the high productivity and sustainable nature of these systems (Madadi et al., 2021). Thus, after characterizing the biomass grown in photobioreactors using different grape pomace extracts, cellulose was also extracted from the biomass. Fourier-transform infrared spectroscopy (FTIR) analysis revealed significant structural changes (Figure 8). The raw biomass samples (control, Biomass E1, and Biomass E2) exhibited a broad absorption band around 3400, 2920, 1730 and 1650 cm -1. The cellulose extracted from different biomasses displayed well-defined characteristic bands at 3330–3400, 2900, 1428, 1367, 1030 and 897 cm -1. Regarding XRD, all cellulose samples exhibited characteristic diffraction peaks near 2θ ≈ 22°. The intensity and sharpness of this peak varied significantly among samples.

Tetradesmus obliquus culture and biomass production parameters

The 0.2 g/L concentration of grape pomace extract used in this study falls within the range reported for bioactive extracts and biostimulants supplemented to microalgal culture media. Pyroligneous acid, a phenolic-rich byproduct chemically comparable to plant pomace extracts, was shown to enhance growth in Chlorella sp. and Desmodesmus sp. MAS1 in photobioreactors at an optimal concentration of only 0.01% (v/v), with higher doses becoming inhibitory (Abinandan et al., 2025). Similarly, exogenous phytohormone supplementation in Chlorella pyrenoidosa achieved maximum biomass (1.00 g/L) and lipid productivity at 10.0 mg/L within a tested range of 1.0–20.0 mg/L (Zhou et al., 2024), while kinetin supplementation increased biomass productivity 1.6-fold in Scenedesmus obtusus at 25 µM (Suparmaniam et al., 2021). The 0.2 g/L concentration adopted here therefore lies within the biologically plausible range for growth promotion without inhibitory effects, consistent with the dose–response pattern reported for bioactive supplements in microalgal cultures.

Additionally, it is important to note that the extracts were not characterized before being added to the photobioreactor; thus, the lack of extract characterization prevents a mechanistic interpretation of the results. With that in mind, the growth kinetics observed in all groups align with the widely documented pattern for green microalgae of the genus Tetradesmus cultivated in closed photobioreactors under batch conditions, in which the exponential phase precedes progressive stabilization associated with nutrient depletion and increased cellular self-shading at higher densities (Nedaei and Shokrkar, 2025; Sánchez Mirón et al., 2002). The growth kinetics of Tetradesmus obliquus respond integratively to light and nutrient availability, with strong regulation under nitrogen limitation and CO 2 supplementation (de Farias Silva et al., 2018; Rörig et al., 2024).

The enhanced performance of E2 aligns with reports demonstrating that phenolic compounds present in grape pomace, such as gallic acid, catechin, and quercetin (Abreu et al., 2025), can act as metabolic modulators in microalgae. These compounds stimulate biomass and lipid accumulation by reducing oxidative stress and redistributing carbon precursors without directly affecting photosynthesis (Zou et al., 2023). Consistently, the addition of extracts did not alter the overall cultivation pattern in the photobioreactors. Microwave-assisted extraction with aqueous ethanol, used to prepare E2, preserves highly bioactive phenolic compounds due to moderate temperature conditions and the use of low-toxicity solvents (Jovanović et al., 2022), which may explain its greater compatibility with the metabolism of T. obliquus compared with E1.

The lower performance of E1 may be associated with the generation of inhibitory compounds during acid hydrolysis. Despite the release of fermentable sugars that could potentially support mixotrophic growth (Çakır et al., 2024; Manso et al., 2025), compounds such as furfural and HMF are known to impair cellular metabolism (Serrano et al., 2023). Moreover, the hydrolysate concentration applied in this study was substantially lower than concentrations commonly reported for efficient mixotrophic cultivation. Previous studies demonstrated that green microalgae such as Chlorella vulgaris achieve robust growth in hydrolysates containing 5–20 g L -1 of organic carbon (Abreu et al., 2012; Lau et al., 2014), reaching biomass values above 3 g L -1. Similarly, the use of agro-industrial residue hydrolysates at high proportions (25%–100% v/v) supports growth rates superior to strictly autotrophic cultivation (Arora and Philippidis, 2021).

The progressive increase in pH observed in all treatments reflects continuous photosynthetic consumption of dissolved CO 2, which reduces carbonic acid concentration and shifts the carbonate equilibrium toward alkalinity (Sforza et al., 2018). The absence of significant differences among treatments indicates that extracts E1 and E2 did not alter the photosynthetic activity rate of T. obliquus at the tested concentration. Similar behavior has been described in the literature, where pH elevation in T. obliquus cultures correlates directly with photosynthetic activity and OH − release during CO 2 assimilation (James et al., 2023; Plöhn et al., 2021), regardless of supplementation conditions (Ferreira et al., 2023).

However, pH values above 8.5 in the final cultivation stages likely imposed physiological limitations on growth. Under alkaline conditions, the equilibrium between dissolved CO 2 and bicarbonate shifts strongly toward HCO 3 −, reducing the fraction of free CO 2 available for RuBisCO carboxylation and consequently limiting photosynthetic carbon fixation (Barceló-Villalobos et al., 2019). This mechanism provides a plausible explanation for the stabilization of cell density after day 26 in all groups.

Overall, the results indicate that extract E2 acted as a low-intensity positive growth modulator without altering the predominant metabolic regime, whereas extract E1 did not promote growth, likely due to the extraction method and concentration applied. These findings demonstrate that the efficiency of agro-industrial residue utilization in T. obliquus cultivation depends critically on the extraction method and applied concentration, which determine the balance between metabolic stimulation and physiological limitation.

Physicochemical characterization

The preparation of extract E1 involved acid treatment, an aggressive method designed to disrupt the lignocellulosic matrix of grape pomace and release fermentable sugars such as glucose and xylose, which can support mixotrophic growth in microalgae (Çakır et al., 2024). The availability of these sugars in the culture medium did not appear to inhibit photosynthetic pigment synthesis, since chlorophyll levels remained comparable to those of the autotrophic control. Similar observations have been reported for mixotrophic systems in which organic carbon supplementation supports growth without suppressing chlorophyll biosynthesis (Arora and Philippidis, 2021).

In contrast, the significant reduction in chlorophyll a and b contents observed in biomass E2 suggests that compounds present in the microwave-assisted ethanolic extract negatively affected pigment accumulation. Polyphenol-rich extracts and/or residual ethanol may create unfavorable conditions for carotenoid and chlorophyll biosynthesis (Li et al., 2024). Although microalgae commonly increase carotenoid production under stress conditions such as high light intensity or nutrient limitation as part of a photoprotective response (Faraloni et al., 2021), the chemical stress associated with E2 supplementation did not promote an increase in β-carotene content. This indicates that the stress imposed by E2 may not have activated classical oxidative stress-response pathways related to carotenoid accumulation.

The stability of carbohydrate content across all treatments suggests that the central metabolic pathways responsible for structural and storage carbohydrate synthesis were not substantially altered by the addition of grape pomace extracts. Although microalgae can assimilate organic carbon directly and often increase biomass productivity under mixotrophic conditions, carbohydrate accumulation is generally less sensitive to organic carbon supplementation than other macromolecules (Chaudhuri and Balasubramanian, 2025).

The elevated protein content observed in biomass E1 indicates that the acid hydrolysis process enhanced nutrient availability in the culture medium. Acid pretreatment of lignocellulosic biomass promotes depolymerization of lignin and hydrolysis of hemicellulose, releasing not only sugars but also organic nitrogen compounds and minerals (Brienza et al., 2024). The higher availability of these metabolic precursors likely favored protein biosynthesis, resulting in a biomass richer in protein. Similar findings have demonstrated that nutrient availability, particularly nitrogen and phosphorus, plays a central role in regulating microalgal metabolism and biochemical composition (Sun et al., 2018; Çakirsoy et al., 2022). The higher protein accumulation in E1 is also consistent with studies reporting improved nutritional quality of microalgal biomass cultivated in agro-industrial residue-based media (Markou and Nerantzis, 2013; Barbera et al., 2018).

The reduction in ash content observed in biomass E1 compared with the control may reflect more efficient mineral uptake by the microalgae in the bagasse-derived medium or differences in mineral composition relative to BBM medium, which contains defined inorganic salts (Markou and Nerantzis, 2013). The even lower ash content detected in biomass E2 suggests that microwave-assisted ethanolic extraction solubilized fewer inorganic minerals from grape pomace. This extraction technique preferentially recovers compounds according to solvent polarity and dielectric properties (Lozano Pérez et al., 2024), which may have reduced the availability of inorganic elements in the culture medium. Consequently, the microalgae may have incorporated lower amounts of minerals into the biomass due to limited nutrient availability (Vítová and Mezricky, 2024).

The predominance of methyl linolenate (C18:3) and methyl linoleate (C18:2) in the control biomass is characteristic of microalgae belonging to the Chlorophyta group, which are typically rich in polyunsaturated fatty acids (Davoodbasha, 2018). The presence of methyl 4,7,10,13-hexadecatetraenoate (C16:4), a short-chain PUFA associated with chloroplast membranes, further indicates active photosynthetic metabolism and chloroplast functionality (Degraeve-Guilbault et al., 2017).

The lower total fatty acid concentration observed in biomass E1 suggests that acid hydrolysis-derived supplementation altered lipid metabolism. The acid treatment applied to grape pomace promotes the release of carbohydrates and phenolic compounds, generating a sugar-rich fraction that may support mixotrophic growth (Serrano et al., 2023). Under mixotrophic conditions, the simultaneous use of light and organic carbon can redirect carbon fluxes and modify lipid biosynthesis pathways, resulting in changes in fatty acid composition (Zeng et al., 2016). In this context, the reduction in major PUFAs such as C18:3 and C18:2, combined with the increase in C16:4, may reflect metabolic adjustments associated with altered carbon availability and nutritional stress, both of which are known to influence lipid accumulation and fatty acid profiles in microalgae (Chandra et al., 2015).

The fatty acid profile of biomass E2, which remained similar to that of the control, indicates that microwave-assisted ethanolic extraction produced a supplement that exerted limited interference on lipid metabolism. Microwave-assisted extraction with ethanol is recognized for its efficiency in recovering polyphenols from grape pomace (Trossolo et al., 2025). These compounds can act as metabolic modulators in microalgae, influencing lipid accumulation and favoring PUFA synthesis when supplied at appropriate concentrations (Zou et al., 2023). The maintenance of high proportions of C18:3 and C16:0 in E2 suggests that the extract preserved metabolic conditions compatible with normal photosynthetic and lipid biosynthetic activity, while still promoting subtle biochemical modulation.

The total fatty acid concentrations obtained in this study (22.409 mg/g for the control and approximately 18 mg/g for the treatments, corresponding to 1.8%–2.2% of dry biomass) were markedly lower than the lipid contents typically reported for T. obliquus in the literature, which usually range from 15% to 40% of dry weight. This discrepancy can largely be attributed to the absence of a deliberate nutrient-limitation step in the present cultivation protocol. High lipid contents in this species are consistently associated with imposed abiotic stress conditions rather than with nutrient-replete growth: under nitrogen deficiency, Scenedesmus (Tetradesmus) obliquus lipid content increased from 12.7% under control conditions to 43% of dry cell weight after cells were transferred to nitrate-deficient medium (Mandal and Mallick, 2009).

This pattern reflects a broader physiological trade-off described in green microalgae, whereby lipid accumulation is induced by stress conditions that compromise photosynthetic activity and growth rate, redirecting photosynthetically fixed carbon away from biomass and structural components toward storage lipids; under nutrient-replete conditions, carbon is instead preferentially allocated to growth and structural biosynthesis. Since the present study employed standard BBM without any nitrogen-limitation or other deliberate stress phase, the low lipid yield is consistent with this carbon-partitioning behavior. In addition, a methodological factor may have contributed to the low values reported here: T. obliquus possesses a rigid, trilaminar cell wall that is highly resistant to solvent and enzymatic disruption, and incomplete cell disruption is a well-documented source of underestimation in microalgal lipid quantification, since solvent-based extraction methods can underestimate fatty acid content when applied to insufficiently pre-treated, rigid-walled biomass (Ghasemi Naghdi et al., 2014).

Regarding minerals, the higher mineral accumulation observed in biomass E1 may be associated with the acid extraction process performed with H 2 SO 4 at 90 °C. This treatment likely promoted extensive disruption of the lignocellulosic matrix of grape pomace, enhancing the release and solubilization of mineral elements and consequently increasing the bioavailability of micronutrients such as Cu, Zn, and Na. The greater availability of these elements may have facilitated their uptake by the microalgae. This interpretation is consistent with the known capacity of microalgae to adsorb and internalize metal ions through surface binding and intracellular transport mechanisms (Cavalletti et al., 2022).

In contrast, the hydroalcoholic microwave-assisted extraction used for E2 tends to selectively recover organic compounds, especially phenolic substances, while limiting the release of tightly bound minerals. As a result, biomass E2 exhibited a comparatively milder mineral profile, despite presenting the highest K concentration among the treatments.

The extraction strategy played a central role in determining the chemical composition of the extracts and their subsequent effects on microalgal cultivation. The higher Fe concentration observed in the control biomass likely reflects the preservation of baseline nutrient availability in the BBM medium. Iron is essential for photosynthetic electron transport and cellular metabolism, and its uptake and homeostasis are tightly regulated in microalgae (Lampe et al., 2024).

From a physiological perspective, the observed differences in mineral composition may significantly influence microalgal metabolism. Elements such as Cu and Zn function as cofactors of redox enzymes, including superoxide dismutase, and therefore participate directly in antioxidant defense systems. However, excessive concentrations of these metals may stimulate the formation of reactive oxygen species (ROS), inducing oxidative stress and triggering metabolic adjustments (Cavalletti et al., 2022). In addition, the elevated Na content observed in biomass E1 may have contributed to ionic imbalance, potentially intensifying stress conditions. Environmental factors such as metal exposure and changes in ionic composition are known to activate antioxidant and adaptive responses in microalgae (Gauthier et al., 2020).

Overall, grape pomace demonstrates strong potential as a value-added agro-industrial residue, particularly because of its content of macronutrients such as potassium (K) and manganese (Mn), which can directly influence the growth and metabolism of Tetradesmus sp. (Machado et al., 2024). These elements are associated with osmotic regulation, enzymatic activation, photosynthesis, and oxidative metabolism, reinforcing their relevance as functional components in microalgal cultivation systems (Zhu et al., 2025). In contrast, biomass E1 was characterized by elevated concentrations of micronutrients such as Cu, Zn, and Na, suggesting that the acid treatment enhanced mineral bioavailability and stimulated uptake and accumulation by the microalgae.

These variations in mineral composition may have important implications for nutritional, biotechnological, and industrial applications, while also reflecting distinct metabolic adaptations under the tested cultivation conditions. Collectively, the findings reinforce the potential of grape pomace valorization within an integrated biorefinery framework (Sirohi et al., 2020; Prata et al., 2025).

Antioxidant activities and total phenolic compounds

The interpretation of antioxidant activity results must consider that the extracts were obtained using methanol, a solvent that preferentially solubilizes polar to semi-polar secondary metabolites. Thus, the antioxidant profile evaluated in this study likely reflects mainly phenolic compounds (e.g., phenolic acids and flavonoids), along with other moderately polar metabolites, while more hydrophobic antioxidants such as carotenoids may be underrepresented (Stalikas, 2007; Plaza et al., 2009).

It is important to emphasize that conclusions regarding antioxidant activity should not be based on a single assay, but rather on a comprehensive evaluation of results obtained from multiple in vitro methods used to measure antioxidant activity (Yang et al., 2023). The results obtained demonstrate that the antioxidant activity of the samples depends on the analytical method employed, reflecting the complexity of the compound profile present and their different mechanisms of action.

The higher DPPH scavenging activity observed in E1 and E2 suggests an increased presence of compounds with strong hydrogen atom donation capacity, typically associated with low-molecular-weight phenolics. In contrast, the superior ABTS activity in the control indicates a broader spectrum of antioxidants, as this assay detects both hydrophilic and lipophilic compounds and is less affected by steric hindrance (Pior et al., 2005). The enhanced FRAP response in E2 further suggests an increased reducing power, reflecting a higher abundance of electron-donating compounds, such as conjugated phenolics or quinone-type structures (Munteanu and Apetrei, 2021).

Moreover, the presence of phenolic compounds in the culture medium may have acted as a chemical stressor, triggering metabolic reprogramming in the microalgae and selectively enhancing the biosynthesis of specific antioxidant metabolites via pathways such as the shikimate and phenylpropanoid routes. Such stress-induced modulation of secondary metabolism has been widely reported in microalgae and is often associated with shifts in both the composition and functionality of antioxidant compounds (Goiris et al., 2015; Yang et al., 2023).

Cellulose extraction

Cellulose recovery from T. obliquus biomass holds particular relevance within the microalgal biorefinery context due to the distinctive architecture of its cell wall. Unlike Chlamydomonas reinhardtii, whose wall is primarily composed of hydroxyproline-rich glycoproteins, T. obliquus possesses a trilaminar structure consisting of an outer trilaminar sheath, an intermediate fibrous cellulosic layer, and a denser inner mature wall adjacent to the plasma membrane (Carmo Cesário et al., 2022; Oliveira et al., 2021). This cellulosic layer confers substantial mechanical resistance to cell disruption, which directly affects the efficiency of downstream extraction of lipids, pigments, and proteins and therefore represents both a processing barrier and a co-product of biotechnological interest (Oliveira et al., 2021). In comparison with conventional lignocellulosic feedstocks, microalgal cellulose offers a processing advantage, since microalgae lack lignin, the recalcitrant aromatic polymer responsible for the costly and environmentally intensive pretreatment steps typically required to isolate high-crystallinity cellulose from plant biomass (Margellou et al., 2024). This positions cellulose recovery from T. obliquus as a complementary, value-added stream within an integrated microalgal biorefinery.

In that way, the raw biomass samples (control, Biomass E1, and Biomass E2) exhibited a broad absorption band around 3400 cm -1, which indicated the presence of hydroxyl groups, as well as signals near 2920 cm -1 corresponded to the (-CH) groups stretching vibration (Hamed et al., 2016). The band around 1730 cm -1 is associated with C=O stretching of ester groups, typically related to lipids and acetylated polysaccharides, while bands near 1650 cm -1 can be attributed to adsorbed water and/or protein contributions. These assignments are consistent with FTIR analyses of complex biomass systems containing mixed biopolymers (Liu et al., 2021).

The cellulose extracted from different biomasses displayed well-defined characteristic bands at 3330–3400 cm -1 (O–H stretching), 2900 cm -1 (C–H stretching), 1428 cm -1 (CH 2 bending associated with cellulose crystallinity), 1367 cm -1 (C–H deformation), 1030 cm -1 (C–O stretching), and 897 cm -1, was attributed to glycosidic bonds of monosaccharides, which is considered a diagnostic band of cellulose (Vârban et al., 2021). These spectral features are in agreement with reported FTIR signatures of cellulose and lignocellulosic materials (Liu et al., 2021; Vârban et al., 2021).

Regarding XRD analysis, sharper and more intense diffraction peaks are generally associated with higher crystallinity due to increased ordering of cellulose chains and the removal of amorphous components (Hemida et al., 2023). The crystallinity index (CrI), calculated using the Segal method, was 36.73% for sample E1, 25.64% for sample E2, and 24.44% for the control. These percentages can be explained by the abiotic and chemical stress, which is known to trigger reinforcement of the microalgal cell wall through transcriptional upregulation of cellulose biosynthesis genes. Similar stress-induced cell wall thickening has been reported across several microalgal genera (Yap et al., 2016), supporting the hypothesis that exposure to a bioactive, phenolic-rich extract such as grape pomace could act as a chemical elicitor of similar defense responses in T. obliquus.

Additionally, phenolic compounds and tannins, which are abundant in grape pomace, are well known to interact directly with cell wall polysaccharides through cross-linking and to be deposited locally as part of the plant defense response, reinforcing the structural matrix and favoring a more ordered, crystalline arrangement of cellulose microfibrils (Kaur et al., 2022). The acid hydrolysis used to obtain biomass E1 may have provided an additional, stronger stimulus for cell wall reinforcement than the predominantly phenolic compounds of biomass E2, in which the microwave-ethanol extraction selectively recovers free phenolics and anthocyanins without generating comparable amounts of polysaccharide-degradation products (Drevelegka and Goula, 2020).

The E1 sample exhibited the most intense and narrow diffraction peak at approximately 22°, indicating the highest degree of crystallinity among the evaluated samples. This result suggests that the extract added to biomass E1 was highly effective in removing amorphous components while preserving or enhancing the crystalline domains of cellulose. Similar behavior has been reported for chemically treated cellulose samples, where increased peak intensity is associated with improved structural ordering (Raza et al., 2024).

The E2 sample also displayed a distinct diffraction peak at approximately 22°, although with lower intensity and a broader profile than E1. Consistent with its intermediate crystallinity index (25.64%), this finding indicates a moderate degree of structural organization and suggests that the extraction process was effective but less efficient than that observed for E1. The presence of additional minor peaks may indicate residual inorganic or non-cellulosic constituents. In contrast, the cellulose extracted from the control biomass exhibited a broader and less intense diffraction peak, together with a higher background signal. The lower crystallinity index (24.44%) confirms the predominance of amorphous regions and suggests less effective removal of non-cellulosic materials. As reported in the literature, untreated or weakly treated biomass generally exhibits broader diffraction peaks due to the coexistence of crystalline and amorphous domains (Hemida et al., 2023).

Overall, the XRD results demonstrate that the extracts used during cultivation significantly influenced the structural organization of the cellulose obtained. The higher crystallinity observed for E1 indicates a greater proportion of ordered cellulose domains, whereas E2 showed only a modest improvement compared with the control. These findings highlight the strong influence of the cultivation treatment on cellulose quality.

In this study, it was demonstrated that grape pomace is an effective substrate for T. obliquus cultivation, influencing both growth dynamics and metabolic composition. All treatments showed typical growth kinetics, with an exponential phase between days 5–10 and stabilization after day 26, likely associated with CO 2 limitation due to pH increase above 8.5. The microwave-assisted extract (E2) promoted higher cell density, while acid hydrolysis (E1) induced a stronger metabolic shift and lower saturated fatty acids. E1 also enhanced protein and mineral contents, whereas antioxidant responses varied according to the extraction method. In addition, cellulose extracted from the biomass showed differences in structural organization and crystallinity, with E1 presenting the highest crystallinity and the control the lowest. Overall, the results demonstrate that grape pomace-derived extracts influence not only microalgal growth and biochemical composition, but also the structural quality of the cellulose obtained, reinforcing the potential of grape pomace for sustainable microalgal biorefineries.