Integrated cultivation of Euglena gracilis in urban wastewater: pilot-scale evaluation of organic matter removal, carbon capture and biomass valorization

Integrated cultivation of Euglena gracilis in urban wastewater: pilot-scale evaluation of organic matter removal, carbon capture and biomass valorization | 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 Integrated cultivation of Euglena gracilis in urban wastewater: pilot-scale evaluation of organic matter removal, carbon capture and biomass valorization Betina Mariela Barreto, Maurício Kersting, Carlos Eduardo Flores Santos, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8949330/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study evaluated the cultivation of Euglena gracilis in real urban wastewater as a strategy for organic matter removal, carbon capture, and biomass valorization. Cultivation was conducted at laboratory and pilot scales using different proportions of urban wastewater and a mineral nutrient medium as control. E. gracilis successfully adapted to the wastewater from a university wastewater treatment plant, showing growth kinetics at pilot scale comparable to laboratory-scale systems. High removal efficiencies were achieved for chemical oxygen demand (up to 98.0%) and ammoniacal nitrogen (up to 91.6%), demonstrating the potential of this organism for organic load reduction. Cultivation in undiluted wastewater enhanced carbon incorporation into the biomass, reaching values above 43%, indicating strong carbon assimilation under mixotrophic conditions. The biochemical composition of the biomass varied according to the culture medium, with a clear metabolic shift from protein-rich biomass in mineral medium to carbohydrate accumulation (up to 40.8%) in wastewater-based cultures. These results demonstrate the feasibility of integrating E. gracilis cultivation into urban wastewater systems at pilot scale, combining organic matter removal with carbon capture and the generation of biomass with biotechnological potential. Euglena gracilis urban wastewater phycoremediation biomass valorization circular bioeconomy carbon capture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The advance of urbanization and industrialization, combined with population growth, has led to a significant increase in the generation of urban wastewater (Sadigov 2022 ). When inadequately managed, these effluents cause severe environmental impacts, such as the eutrophication of water bodies and the degradation of ecosystems (Li et al. 2023b ; Hait et al. 2024 ). This scenario highlights the urgent need to develop efficient and sustainable methods for wastewater treatment, making this issue a global priority due to the necessity of mitigating environmental impacts and preserving water resources (Khan et al. 2022 ; Obaideen et al. 2022 ). Recent studies have demonstrated the effectiveness of physicochemical and hybrid approaches based on iron-derived materials for wastewater treatment. Falyouna et al. ( 2022 ) reported that zero-valent iron nanoparticles deposited on titanium nanowires achieved high adsorption efficiency for the removal of ciprofloxacin from aqueous solutions, highlighting the importance of nanostructured supports. Eljamal et al. ( 2024 ) showed that the combined use of iron nanoparticles and Aloe vera biomass significantly enhanced biogas production and process stability during anaerobic digestion of waste sludge. In addition, Maamoun et al. ( 2023 ) demonstrated effective removal of phosphorus and nitrate from aqueous solutions through statistical optimization of zero-valent iron nanoparticle synthesis. Collectively, these studies indicate that iron-based technologies can achieve high contaminant removal efficiencies, although they rely on externally supplied materials and focus primarily on pollutant removal. Other emerging approaches, integrated urban wastewater treatment systems associated with the cultivation of photosynthetic microorganisms have proven to be a promising technological alternative, both for the removal of organic load and for the production of high-value biomass (Morais et al. 2022 ; Cheirsilp et al. 2023 ). Considering the microorganisms studied, E. gracilis stands out as a unicellular euglenoid which, although not belonging to the true microalgae group, exhibits similar photosynthetic metabolism and physiological behavior, being able to grow under autotrophic, heterotrophic, or mixotrophic conditions. This adaptive capacity expands its potential for applications in different types of effluents, making it an ideal candidate for the remediation of contaminated urban environments (Alves et al. 2024 ; Nezbrytska et al. 2022 ). Photosynthetic microorganism-based systems have been increasingly explored within a biorefinery approach for wastewater treatment, as they enable simultaneous nutrient removal and biomass production. These systems are effective in reducing nitrogen and phosphorus concentrations, which are key drivers of eutrophication in aquatic environments, by assimilating dissolved nutrients during biomass growth (Butzke et al. 2024 ; Nezbrytska et al. 2022 ). Technologies such as photobioreactors and high-rate algal ponds (HRAPs) provide favorable conditions for biomass accumulation and wastewater polishing and have demonstrated promising performance at pilot and semi-industrial scales across different climatic regions (Sutherland and Ralph 2020 ). When integrated with physical and biological processes, including sedimentation and activated sludge, these systems can further enhance treatment efficiency while reducing operational costs (González-Camejo et al. 2021 ). Within this biorefinery framework, E. gracilis has received particular attention due to its multifunctional role in wastewater treatment. In addition to contributing to nutrient removal, this euglenophyte is capable of biofixing carbon dioxide during growth, converting inorganic carbon into biomass enriched in compounds of high economic value, which supports greenhouse gas mitigation efforts (Molazadeh et al. 2019 ). Moreover, biomass produced from wastewater-grown E. gracilis has been reported as a suitable feedstock for biofuel production, including biodiesel, biogas, and bio-oil, offering a renewable alternative to fossil energy sources (Toyama et al. 2018 ). The use of microalgal biomass for biofuel production presents advantages such as high biomass productivity and the ability to grow in non-arable or saline environments without the need for irrigation, herbicides, or pesticides (Deviram et al. 2020 ). E. gracilis exhibits unique characteristics that make it particularly attractive for biotechnological applications, being an excellent source of dietary proteins, vitamins, lipids, and β-1,3-glucan (paramylon), a polysaccharide exclusive to euglenoids (Khatiwada et al. 2020 ) Its high rate of bioproduct synthesis under different culture conditions positions it as a viable competitor to microalgae such as those of the Chlorella genus, whose products are already commercially available (Sayar and Eryalçın 2023 ). Regarding the use of this biomass, it is noteworthy that E. gracilis has traditionally been used as food in some Asian countries (Mou et al. 2024 ). In others, it is already recognized as a novel food (Nuin Garciarena et al. 2025 ) or generally recognized as safe (GRAS) (Guo et al. 2025 ). However, when cultivated in urban wastewater, the use of E. gracilis biomass faces restrictions due to the potential presence of contaminants. In such cases, its use is more viable in non-food applications, such as agriculture, biomaterial production, and biofuels (Braga et al. 2022 ; Okeke et al. 2022 ). The application of E. gracilis in wastewater treatment has been associated with efficient nutrient removal while enabling the production of biomass enriched in compounds such as proteins, lipids, and pigments, thereby increasing its biotechnological relevance (Butzke et al. 2024 ). Its integration into effluent treatment systems has been proposed as a sustainable strategy to mitigate the environmental impacts of wastewater discharge while supporting resource recovery (Nezbrytska et al. 2022 ). Within this context, the use of E. gracilis has been framed under a biorefinery concept, in which wastewater serves not only as a treatment target but also as a cultivation medium for generating value-added bioproducts (Khatiwada et al. 2020 ). The incorporation of E. gracilis cultivation into wastewater treatment processes may also contribute to improved process efficiency and reduced operational constraints, while enabling effluent valorization through biomass utilization (Toyama et al. 2018 ; Barsanti et al. 2020 ). Overall, this multifunctional approach combines environmental remediation with the sustainable production of high-value bioproducts, reinforcing the importance of developing integrated and efficient technologies for water and resource management (Wu et al. 2021 ; Molinuevo-Salces et al. 2019 ). Previous studies have demonstrated the feasibility of cultivating Euglena spp. in domestic wastewater, primarily at laboratory scale or under controlled conditions. At lab scale, Euglena sp. has been shown to grow mixotrophically in untreated domestic sewage, achieving efficient removal of organic matter and nutrients such as nitrogen and phosphorus, while producing biomass with relevant lipid content for biorefinery applications (Mahapatra et al. 2013 ). More recent investigations have explored mixed cultures, showing that E. gracilis combined with other microalgae, such as Selenastrum sp., can enhance wastewater treatment performance. In aquaculture wastewater systems, mixed cultures of E. gracilis and Selenastrum achieved near-complete ammonium and phosphate removal, along with high reductions in total nitrogen and phosphorus and increased biomass and lipid productivity compared to monocultures (Tossavainen et al. 2019 ). In addition, the ability of Euglena to adapt to real wastewater streams has been demonstrated in engineered systems, such as sequencing batch membrane photobioreactors fed with secondary effluents, where native Euglena strains achieved high nutrient removal efficiencies while producing lipid-containing biomass under mixotrophic conditions (Sheng et al. 2017 ). At pilot scale, studies involving Euglena in domestic wastewater have been more limited and have largely focused on its natural occurrence within complex algal–bacterial communities rather than on its targeted application. In pilot-scale attached-growth pond systems treating raw domestic wastewater, Euglena was identified as one of the dominant algal groups contributing to organic matter and nutrient removal (Phyu et al. 2024 ). Similarly, long-term pilot-scale multimodal algal–bacterial processes treating municipal wastewater have consistently detected members of the Euglenophyceae, including Euglena spp., particularly under mixotrophic conditions, where they contributed to system resilience and nutrient assimilation despite not being the dominant taxa (Mahapatra and Murthy 2021 ). Although these studies highlight the ecological relevance and adaptability of Euglena in pilot-scale wastewater treatment systems, they also reveal a clear gap in the literature: systematic investigations involving the deliberate inoculation of E. gracilis into raw domestic wastewater at pilot scale, coupled with an integrated evaluation of growth kinetics, treatment performance, carbon assimilation, and biomass composition, remain scarce. This gap contrasts with the recognized biotechnological potential of E. gracilis within biorefinery-oriented wastewater remediation frameworks (Khatiwada et al. 2020 ) and directly motivates the need for the present pilot-scale study. Therefore, this study aimed to assess the potential of E. gracili s for the treatment of urban wastewater, evaluating its feasibility for biomass production with biotechnological applications. In this context, it represents an innovative approach by employing E. gracilis in urban effluent treatment, simultaneously exploring its capacity for nutrient removal, carbon biofixation, and the generation of biomass with biotechnological value. The use of this microorganism in wastewater represents a significant advancement over conventional systems by transforming an environmental liability into a source of renewable resources. Hence, it reinforces the importance of integrating environmental biotechnology with the principles of the circular economy, highlighting promising pathways for the development of clean and sustainable process technologies. 2. Materials and Methods 2.1 Cultivation E. gracilis , registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under number A863DC8, was used in this study The urban effluent employed was collected from the Wastewater Treatment Plant (WWTP) of the University of Santa Cruz do Sul (UNISC). After sampling, the effluent stood for 10 minutes to allow the sedimentation of suspended solids, and the pH was adjusted using a 5% phosphoric acid (H₃PO₄) solution, considering that E. gracilis exhibits optimal growth under acidic conditions (Xie et al. 2023 ). This adjustment also helped to minimize contamination by other microorganisms, favoring the predominant development of the target species. As a reference medium, an NPK (12:11:18) solution at a concentration of 3 g L⁻¹ was used, which had been previously employed in the maintenance cultures of the strain and served both for inoculum production and as the control condition in the experiments (Alves et al. 2024 ). Laboratory-scale cultures were carried out in photobioreactors with a working volume of 4 L, inoculated with E. gracilis at 20% of the total volume. The system was maintained for up to three weeks under a photoperiod of 12 hours of light and 12 hours of darkness, with artificial illumination provided by 20.5 W tubular LED lamps (average of 148 µmol photons m⁻² s⁻¹), in an environment without temperature control. Aeration was provided through a bubble column generated by an air compressor (VigoAr 200 Plus), using a porous stone to ensure homogeneous air distribution. Different proportions of real raw urban wastewater were evaluated, corresponding to 10, 20, 30, 40, and 100% (v/v) of the culture medium. Considering the use of 20% inoculum and the addition of effluent percentages, the final volume was completed with NPK solution (3 g L⁻¹) according to the effluent proportion tested in each experimental condition. At the pilot scale, cultivation was conducted using 100% raw urban wastewater in 100 L plastic bags equipped with a bubble column, maintained indoors under artificial illumination and a 12:12 photoperiod, as illustrated in Fig. 1 . The cultivation system consisted of plastic-bag photobioreactors made of low-density polyethylene (LDPE) smooth plastic bags (220 × 40 cm; Crudo Plast), arranged vertically and illuminated by LED panels that uniformly illuminated the entire set of bags. Aeration was provided by a high-flow air compressor (Air 140 compressor for ornamental ponds, 220 V, 140 L min⁻¹), generating a continuous bubble column, with airflow limited by the size of the air inlet orifice (1 cm) into the bag. Temperature and irradiance were recorded using a HOBO data logger (UA-002-64, Onset Computer Corporation, USA). 2.2 Monitoring of Cell Growth The development of the cultures was monitored weekly through cell counting in a hemocytometer using a microscope and spectrophotometric analyses performed with a UV–Vis spectrophotometer, with readings at 680 nm — the wavelength corresponding to the maximum absorption of chlorophyll — used as an indicative parameter of cell growth. The equipment was calibrated using distilled water as a blank. In parallel, the pH of the samples was measured with a benchtop digital pH meter, allowing the monitoring of variations in this parameter throughout the experiment. At each monitoring interval, samples were collected to determine biomass, which was separated by centrifugation and subsequently dried to constant weight. The calculation of cells per mL was performed based on Eq. 1, using counts obtained with a Sedgewick–Rafter counting chamber, where N is the number of cells counted, A is the field area in mm² (1), D is the chamber depth in mm (1), and F is the number of fields counted (10). For the calculation of kinetic data referring to the specific growth rate (µ), the generation rate (k), and the doubling time (td), Equations 2 to 4 were used, where x₁ and x₂ are the initial and final cell counts (cells mL⁻¹) at times t₁ and t₂ (days). \(CellD\left(cell{mL}^{-1}\right)=\frac{Nx1000{mm}^{3}}{AxDxF}\) Eq. 1 \(\mu=\frac{lnx2-lnx1}{t2-t1}\) Eq. 2 \(k=\frac{\mu}{ln2}\) Eq. 3 \({t}_{d}=\frac{ln2}{\mu}\) Eq. 4 Culture monitoring was also carried out through physicochemical parameters, including Dissolved Oxygen (DO), Electrical Conductivity (EC), and Total Suspended Solids (TSS), measured using a multiparameter meter (AK-88, Akso, Italy); turbidity, measured with a digital turbidimeter (AK410 – Turbidez Max, Akso, Italy). 2.3 Effluent Parameter Testing Samples were previously centrifuged and filtered through 0.45 µm membranes to remove suspended particles and subsequently diluted in ultrapure water to ensure the accuracy of chemical component measurements. The methods described for Chemical Oxygen Demand (COD), SMEWW 5220 B, and Ammoniacal Nitrogen (N–NH₃), SMEWW 4500–NH₃ B, were employed according to Standard methods for the examination of water and wastewater (APHA 2017 ). The parameters of Total Organic Carbon (TOC), Total Inorganic Carbon (TIC), and Total Nitrogen (TN) were determined using a TOC/TN elemental analyzer (TOC-LCPH/CPN, Shimadzu, Japan). 2.4 Biomass Characterization Final biomass separation was performed by centrifugation using a refrigerated benchtop centrifuge operating at 3200 rpm for 5 minutes. After this procedure, the final supernatant was collected and reserved for physicochemical analyses, while the biomass pellet was subjected to lyophilization in a benchtop freeze dryer (LS3000, Terroni, Brazil) for preservation and subsequent characterization. The dried biomass was evaluated for gravimetric yield and biochemical composition, focusing on biomolecules of interest, including lipids, proteins, and carbohydrates. Lipid content was determined using the method adapted from Bligh and Dyer ( 1959 ), as recently applied by Butzke et al. ( 2024 ). Protein content was estimated from nitrogen content determined by elemental analysis (N × 4.78), while the fixed carbon content corresponded to the percentage of total carbon measured in the biomass (Martini et al. 2019 ; Templeton and Laurens 2015 ). Carbohydrate quantification followed the protocol described by Sluiter et al. ( 2010 ), using the concentrated acid hydrolysis method followed by analysis by high-performance liquid chromatography with refractive index detection (HPLC/RID, 20A, Shimadzu, Japan). 2.5 Statistical Analysis Results were expressed as mean ± standard deviation, based on descriptive data analysis. Statistical analysis was performed using GraphPad Prism software (version 10.5.0). Data were initially tested for normality and homogeneity of variances to assess the suitability of parametric analyses. When these assumptions were met, differences among cultivation conditions were evaluated using one-way analysis of variance (ANOVA), followed by appropriate post hoc tests, with statistical significance set at p < 0.05. Statistically significant differences between means are indicated by different letters. When normality assumptions were not met, data were analyzed using the non-parametric Kruskal–Wallis test followed by Dunn’s post hoc test. In cases where graphical complexity or limited replication precluded the inclusion of statistical lettering in figures or tables, statistically significant differences (p < 0.05) are reported directly in the text. For infrared spectral analysis of the biomass, the ChemoStat 4 software was used. Principal component analysis (PCA) was applied to the mean of the normalized spectra, with data preprocessed by mean-centering. 3. Results and Discussion 3.1 E. gracilis Growth As shown in Table 1, all conditions with the addition of urban effluent exhibited cellular growth. After 21 days, the following order of cell accumulation was observed: 40% > 30% ≈ 20% > 10%. During the first week of cultivation, the greatest increase in biomass was recorded, particularly in the condition with 100% effluent addition, indicating higher nutrient bioavailability at the beginning of cultivation. This behavior is in line with that described by Liao et al. (2024), who reported that higher nutrient loads during the initial stages favor nitrogen and phosphorus assimilation by microalgae, promoting more intense exponential growth rates until the gradual depletion of available nutrients. The use of 100% effluent allowed for biomass production within 14 days equivalent to that obtained in 21 days under conditions with 10–40% effluent, due to a faster growth kinetic (shorter cell doubling time). However, in denser or deeper cultures, factors such as self-shading, light limitation, optical interference of the effluent (color, turbidity), possible inhibitors present in the medium, and aeration constraints may attenuate this kinetic advantage and limit effective growth (Saccardo et al. 2022). Even considering factors that may interfere with growth, among the dilutions tested, the 40% concentration showed the highest increase in cell concentration. These results highlight the feasibility of using effluent as a cultivation medium for E. gracilis , as also reported by Butzke et al. (2024), who achieved similar results using swine effluent with the same E. gracilis strain. However, higher concentrations of swine effluent did not yield better results, unlike what was observed with 100% urban effluent. The use of effluent concentrations between 10% and 40% resulted in biomass yields ranging from 0.43 to 0.69 g L⁻¹. The dry biomass yield varied according to the different cultivation conditions, demonstrating the adaptability of E. gracilis to urban effluent. In the 100% effluent condition, the biomass obtained was closer to the value achieved using NPK as the nutrient source. However, in the pilot-scale system, harvesting must occur earlier—or the culture medium must be partially renewed—since after 11 days the biomass density had already entered a decline phase. Table 1 Values of µ in day⁻¹, td in days, k in divisions per day for E. gracilis in medium with effluent compared to NPK. Scale Wastewater content µ (d − 1 ) Maximum cultive days t d k (d − 1 ) Biomass (g L − 1 ) Bench 10% 0.039 21 17.76 0.056 0.43 20% 0.051 21 13.57 0.074 0.69 30% 0.052 21 13.32 0.075 0.55 40% 0.056 21 12.43 0.080 0.56 NPK 0.162 14 4.29 0.230 0.77 100% 0.108 15 6.39 0.156 0.65 Pilot NPK 0.118 14 4.53 0.225 0.93 100% 0.156 11 6.12 0.222 0.40 * The data were calculated considering experiments conducted during the same weeks; therefore, they were not performed in triplicate. The growth of E. gracilis in raw effluent was monitored through cell density (CellD) and optical density, as shown in Fig. 2, for the culture in 100% effluent. The results indicate that E. gracilis was able to assimilate the available nutrients without the need for dilution, demonstrating its potential for the bioremediation of wastewater. Monitoring carried out with the multiparameter probe also revealed significant changes (p < 0.05) in medium quality: color, measured by absorbance at 660 nm, increased from 0.229 ± 0.105 to 0.904 ± 0.099, following the rise in biomass density; EC decreased from 699.6 ± 2.7 to 162.4 ± 0.4 mS; TSS decreased from 350.3 ± 1.3 to 81.9 ± 0.2; and DO increased from 2.8 ± 1.0 to 6.2 ± 0.9 mg L⁻¹. At the pilot scale, the kinetic parameters indicated behavior similar to that observed at the laboratory scale, though with some variations associated with environmental and operational conditions. During the exponential phase, cultures in 100% effluent and in the NPK medium exhibited specific growth rates (µ) of 0.156 and 0.118 day⁻¹, respectively—values comparable to those obtained at the laboratory scale (0.108 and 0.162 day⁻¹). The observed differences can be attributed to factors such as air bubble distribution, temperature, light incidence, and biomass accumulation at the bottom of the bags, which directly influence the photosynthetic efficiency of E. gracilis . The cell doubling time (td) remained within a similar range between the two scales, being 6.12 days in 100% effluent and 4.53 days in NPK medium, compared to 6.39 and 4.29 days at the laboratory scale, respectively. These results suggest that the pilot system was able to maintain yield comparable to bench scale, even without strict temperature control, as shown by the temperature and irradiance data in Fig. 3. The cell division coefficient (k) also showed little variation between scales, indicating that the adaptation of E. gracilis to the larger-volume system was similar. Figure 3 presents the monitoring of irradiance and temperature throughout the cultivation in the pilot system. Temperature (Fig. 3a) ranged from 24 to 35°C during the experiment, which was conducted between the spring and summer of 2024–2025, a period characterized by high temperatures in southern Brazil. Although the experiments were carried out indoors, the space was not climate-controlled, resulting in significant thermal fluctuations. E. gracilis was able to tolerate this gradient; however, previous studies, including those conducted in our laboratory, indicate that its growth is more favorable under controlled conditions at 25°C. In this context, the lack of temperature control may have acted as a limiting factor in the results obtained. For instance, Wang et al. (2018) observed that E. gracili s reaches its peak specific growth rate between 27 and 30°C, indicating that significant deviations from this range may reduce its cell multiplication efficiency. The recorded irradiance (Fig. 3b) varied from 28 to 40 µmol m⁻² s⁻¹, a range similar to that used by Butzke et al. (2024) during the light period of a 12:12 photoperiod. These values are also consistent with the light intensities recently reported as typical for microalgae production by Vo et al. (2024) and Adetunji et al. (2025). 3.2 Nutrient Removal in the Cultivation of E. gracilis in Urban Effluent The results of the carbon, nitrogen, and phosphorus analysis in the liquid medium after cell separation are presented in Table 2. The 25.9% reduction in TOC indicates the effective removal of organic compounds present in the effluent, reflecting the ability of E. gracilis to assimilate and oxidize organic matter during cultivation. Removal values of this magnitude or higher are typically observed in mixotrophic systems using photosynthetic microorganisms cultivated in urban effluents, especially when good adaptation to organic carbon sources is achieved (Mahapatra et al. 2013; Farjallah et al. 2024). In addition, the synergistic action of heterotrophic bacteria that may be present in the effluent can degrade organic compounds and contribute to TOC removal (Phyu et al. 2024). However, the efficiency of the removal process can be influenced by factors such as light availability, the presence of essential nutrients, and the carbon-to-nitrogen ratio in the medium (Pang et al. 2019). Indeed, Dang et al. (2022) and Xu et al. (2017) demonstrated that inadequate C/N ratios (either too low or too high) result in lower nitrogen and phosphorus removal efficiency, as well as negatively affecting microalgal growth rates. In mixotrophic cultures, Khanra et al. (2020) observed that adjustments in the C/N ratio modulate the assimilation of organic carbon and the protein metabolism of the cells. Similarly, Pereira et al. (2024) reported that variations in the C/N ratio influence the removal of dissolved organic carbon by microalgae. According to Xie et al. (2023), the optimal C/N ratio was 10, and in our system, whose initial C/N ratio was 1.9, it is likely that this value was outside the optimal range for E. gracilis , which may have limited additional TOC removal and the utilization of residual nitrogen. These results indicate that, for the efficient use of this strain in treatment systems, future studies should evaluate the influence of different nutrient proportions on the growth and cellular metabolism of this species. The expressive 99.1% removal of inorganic carbon (IC) indicates the fixation of inorganic carbon by E. gracilis . This result can be explained by the fact that dissolved inorganic carbon represents the main carbon source for photosynthesis in microalgae (Kusi et al. 2024). Additionally, this reduction may be related to the cultivation of E. gracilis as an efficient carbon fixation system. Razzak et al. (2017) highlighted that photobiological systems can remove more than 80% of inorganic carbon from effluents, depending on cultivation conditions such as aeration and light availability. The 69.5% removal of total carbon (TC) reflects the combined contribution of both organic and inorganic carbon assimilation processes. Similar results have been reported in studies that employed microalgae for wastewater treatment, where TC removal ranged from 50% to 80%, depending on effluent composition and experimental conditions (Lu 2025). However, Toyama et al. (2018) noted that the efficiency of TC removal is affected by the presence of symbiotic bacteria, which assist in the conversion of organic matter and promote the release of CO₂ for fixation into the biomass. The increase observed in total nitrogen and total phosphorus concentrations at the end of treatment is related to the addition of the E. gracilis inoculum previously cultivated in synthetic medium containing NPK, which introduced residual nutrients into the system. This effect, however, was less pronounced when an inoculum previously adapted to the effluent was used, indicating that prior adaptation constitutes an effective strategy to minimize external nutrient input during the treatment process. Nevertheless, considering that the culture was initiated with an isolated Euglena strain, supplementation with NPK in the inoculum and pH adjustment were necessary to ensure its predominance in the microbial consortium. In applied systems, longer hydraulic retention times and integration with other nature-based technologies (such as constructed wetlands or high-rate algal ponds) may enhance nutrient removal and reduce the dependence on external input addition (Sutherland and Ralph 2020). It is understood that microalgae can represent one component of a broader treatment system, as they remove emerging contaminants and nutrients, increase dissolved oxygen, reduce chemical oxygen demand, and establish a symbiotic environment with bacteria during the treatment process (Nguyen et al. 2022; Bang Truong et al. 2024; Abdelfattah et al. 2023). The evaluation of treatment efficiency applied to the effluent generated at the university campus demonstrated that the system employing E. gracilis met some of the emission standards established by CONSEMA Resolution No. 355/2017, particularly concerning Chemical Oxygen Demand (COD) and Ammoniacal Nitrogen (N–NH₃). Table 2 Parameters of the effluent before and after treatment with E. gracili s in 100% effluent. Parameters Initial After 3 weeks Removal (%) Carbon Organic 38.92 28.84 ± 2.23 25.9 Inorganic 53.23 0.47 ± 0.10 99.1 Total 93.05 28.36 ± 2.26 69.5 Nitrogen Total 39.45 41.31 ± 17.33 - Ammoniacal 14.4 1.20 ± 0.99 91.6 Phosphorus Total 2.73 55.96 ± 0.83 - Eletrical Conductivity (µScm⁻¹) 1013 793 ± 179 21.7 Chemical Oxygen Demand (COD) (mg L⁻¹) 99,2 ˂2.0 97.9 Turbidity (NTU) 52 3.24 ± 1.9 93.8 Total Suspended Solids (TSS) 350.3 1.3 ± 0.2 99.6 pH 7.6 5* - *pH adjusted for cultivation COD removal reached 98%, a result superior to that obtained in the study conducted by Butzke et al. (2024), who reported a reduction efficiency of 44% in diluted swine wastewater (25%). This difference may be attributed to the higher nutrient concentration in that type of effluent, which initially presented COD values of 3101 mg L⁻¹—substantially higher than the initial values of the raw urban effluent used in the present study. Kim et al. (2021) demonstrated that the cultivation of E. gracilis in tomato processing wastewater under continuous illumination (24 h light) achieved a COD removal of 23% at pH 3. Comparison with these studies indicates that the environmental conditions adopted in the present work—such as the 12:12 light–dark photoperiod and acidic pH—likely favored the optimal utilization of organic carbon fractions present in the wastewater. This emphasizes the importance of environmental conditions for achieving efficient nutrient removal by photosynthetic organisms such as Euglena. It is also important to highlight the remarkable efficiency of the system in the removal of N–NH₃, reaching values close to 97%, corresponding to a final concentration below 0.5 mg L⁻¹. As expected for E. gracilis , ammoniacal nitrogen is primarily removed from the culture medium (He et al. 2025). This performance is comparable to that reported by Silveira et al. (2020) in an integrated system (microalgae associated with constructed wetlands), which achieved 98% nitrogen removal. Kuroda et al. (2018) also demonstrated high nutrient removal potential in primary sedimentation tank effluent, where during 14 days of cultivation E. gracilis almost completely removed ammonium (NH₄⁺–N), resulting in final concentrations below 0.2 mg L⁻¹. 3.3 Biomass Yield and Composition The biochemical composition of E. gracilis biomass varied significantly according to the proportion of urban effluent in the culture medium (Table 4), reflecting metabolic adjustments to different nutrient availability and physicochemical conditions. The results indicate that the highest protein synthesis occurred under control conditions (NPK only), both at pilot scale (40.0%) and laboratory scale (49.9%). These higher values are associated with media rich in readily assimilable inorganic nitrogen, which is essential for the synthesis of structural and enzymatic proteins (He et al. 2025; Yaakob et al. 2021). The progressive addition of effluent resulted in a significant reduction in protein content, especially at the 100% effluent concentration, where the value decreased to 23.9%. Table 3 Biochemical composition of E. gracilis dry biomass cultivated at different concentrations of urban effluent. Wastewater concentration (%) Biochemical composition (%) Lipids Protein Carbohydrate Pilot-scale Control (only NPK) 7.3 ± 0.4 40.7 ± 2.1 13.4 ± 0.0 100 24.4 ± 0.5 27.4 ± 0.7 40.0 ± 0.0 Bench-scale Control (only NPK) 15.7 ± 0.7a 49.9 ± 0.1a 13.8 ± 0.8a 10 22.0 ± 0.9b 35.9 ± 0.1b 9.43 ± 0.1b 20 21.8 ± 2.2b 38.6 ± 0.1b 17.1 ± 0.3b 30 23.9 ± 4.8b 37.6 ± 0.1b 20.8 ± 0.8b 40 23.2 ± 0.7b 37.2 ± 0.1b 20.0 ± 0.2b 100 18.5 ± 1.4a 23.9 ± 2.6c 40.8 ± 1.6c Means followed by the same letter do not differ significantly according to analysis of variance (ANOVA) at p < 0.05. This reduction can be attributed to the higher C/N ratio in urban effluent, characterized by a relative excess of organic carbon and a lower availability of assimilable nitrogen. According to Xie et al. (2023), E. gracilis tends to redirect its metabolism as the C/N ratio increases, reducing protein synthesis while favoring the formation of storage compounds. Therefore, mineral media (NPK) or effluent diluted up to 20% are more suitable when the goal is to maximize the protein content of the biomass. An opposite trend was observed for carbohydrates, whose content increased with the rise in effluent proportion. At the laboratory scale, values ranged from 13.8% (control) to 40.8% (100% effluent), indicating intense carbohydrate accumulation under high organic load. This increase is related to nitrogen limitation and metabolic stress, which induce E. gracilis to accumulate the polysaccharide paramylon, an energy reserve analogous to starch. According to Bakku et al. (2023) and Toyama et al. (2018), this response is typical of mixotrophic or heterotrophic cultures subjected to carbon excess and inorganic nutrient restriction. In the pilot system, the same trend was observed, although less pronounced, suggesting that physical factors such as light dispersion, thermal gradients, and aeration limitation may modulate the biochemical response of the biomass (Farjallah et al. 2024). Thus, the presence of urban effluent significantly altered intracellular carbon allocation, redirecting the metabolism of E. gracilis from active protein synthesis toward the accumulation of energy reserves. The effluent provides a complex mixture of organic and inorganic compounds that modifies nutrient availability, turbidity, and light penetration in the medium, thereby affecting photosynthetic activity (Abdelfattah et al. 2023). In addition, the possible symbiotic activity of heterotrophic bacteria present in the effluent may contribute to the mineralization of organic matter and the release of assimilable CO₂ (Toyama et al. 2018), reinforcing the mixotrophic nature of the culture. These findings corroborate the results reported by Alves et al. (2024) for the same strain cultivated in NPK medium supplemented with a high-organic-load residue, which exhibited low lipid (8.4%) and protein (35.9%) contents and a high carbohydrate content (66.5%). Considering that the same samples used in the laboratory-scale experiments were also analyzed by FTIR, a clear agreement was observed between the results obtained from the different analytical approaches. The characteristic bands of the main compound groups are highlighted in Fig. 4, showing intense signals around 1100 cm⁻¹, attributed to C–O–C and C–O vibrations typical of carbohydrates (Ferro et al. 2019). Bands of lower intensity were also observed near 1650 cm⁻¹, corresponding to the C = O stretching (Amide I) of protein chains, and around 2900 cm⁻¹, associated with the C–H vibrations of methyl and methylene groups present in lipids (Mahapatra et al. 2013; Zhu et al. 2024; Murdock and Wetzel 2009). The spectra were subjected to principal component analysis (PCA), as shown in Fig. 4. The PC1 axis explained 91.27% of the total variance, representing the main source of difference among the samples. This component primarily discriminated the presence of functional groups associated with carbohydrates, since the samples cultivated in medium containing 100% effluent exhibited a higher proportion of these compounds. The PC2 axis explained 5.34% of the variance and reflected variations related to protein content: samples located on the negative side of PC2 and positive side of PC1 exhibited higher protein levels and lower carbohydrate content, except for the sample cultivated in NPK medium, positioned in the positive quadrant of this component, which showed high protein content and lower lipid content. These results corroborate the infrared spectroscopic analyses, confirming through PCA of the spectra the trend observed in the data presented in Table 4. Regarding the proteins of E. gracilis obtained from the different systems tested, it was observed that they are mainly composed of the amino acids arginine, glutamic acid, aspartic acid, and alanine, indicating a protein profile with relevant amounts of essential amino acids, as shown in Fig. 5. These values were similar to those reported by Butzke et al. (2024) for E. gracilis cultivated in swine wastewater for the purpose of producing a germination biostimulant. Most amino acids showed a significant difference (p < 0.05) between the cultivation conditions. Notably, arginine concentration was markedly higher in the biomass obtained from the bench-scale system cultivated with 100% urban wastewater compared to the other conditions, and this amino acid is of particular importance for plant metabolism. This composition reinforces the potential application of the biomass in agricultural bioinput formulations (Xie et al. 2023). On the other hand, cultures grown under conditions that favor carbohydrate accumulation—particularly paramylon—make the biomass a promising source of paramylon nanofibers (Azuma and Yamamoto 2024), which can be hydrolyzed into glucose and subsequently used for bioethanol production. Research has demonstrated that, after hydrolysis and fermentation processes, ethanol can be obtained from E. gracilis biomass (Mou et al. 2024). Considering that the biomass obtained from pilot-scale cultivation in 100% effluent presented an average concentration of 0.58 g L⁻¹ with 40% total carbohydrates, an equivalent fraction of 0.23 g L⁻¹ of carbohydrates was estimated. Of this total, 63.9% correspond to glucose derived from paramylon hydrolysis, resulting in approximately 0.148 g L⁻¹ of glucose available for fermentation. Based on the stoichiometry of glucose conversion to ethanol (C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂) by Saccharomyces cerevisiae , the theoretical ethanol yield is 0.51 g g⁻¹ of glucose, corresponding to a production potential of 0.075 g L⁻¹ of ethanol. Although this value is modest, it demonstrates that the biomass of E. gracilis cultivated in effluent represents an alternative source of fermentable substrate. 3.4 Carbon Capture Carbon capture varied significantly among the evaluated conditions, demonstrating the effect of medium composition and cultivation scale on the metabolism of E. gracilis (Table 4). It was observed that the use of 100% urban effluent promoted the highest carbon incorporation into the biomass, both at the laboratory scale (43.66%) and at the pilot scale (43.33%), surpassing the values obtained in the NPK controls. This behavior indicates that the effluent provided not only inorganic carbon sources but also assimilable organic fractions, which is characteristic of mixotrophic systems. Recent studies have reinforced that the presence of dissolved organic carbon in effluents stimulates carbon assimilation by microalgae, resulting in high net fixation rates and potentially carbon-negative systems due to the simultaneous utilization of atmospheric CO₂ and available organic carbon (Abdelfattah et al. 2023; He et al. 2025; Wang et al. 2024). Table 4 Elemental analysis of carbon captured in the biomass of E. gracilis cultivated in urban effluent medium. Escale Effluent (%) Carbon (%) (mean ± SD) Pilot 100 43.33 ± 0.45a NPK 40.74 ± 0.40b Laboratory 10 38.00 ± 1.91c 20 40.36 ± 2.01b 30 39.54 ± 1.97bc 40 38.84 ± 1.94c 100 43.66 ± 2.10a NPK 38.84 ± 2.19c Carbon capture in microalgal systems is intrinsically linked to biomass growth and carbon allocation within the cells. In this context, the pilot-scale cultivation of E.gracilis in the present study showed stable growth kinetics and consistent incorporation of carbon into the biomass under realistic operational conditions. Similar behavior has been reported in previous pilot and semi-pilot studies with E. gracilis , which demonstrated the capacity of this species to sustain growth and metabolic activity in wastewater-based systems subject to environmental variability (Nezbrytska et al. 2022). Although methodological differences prevent direct quantitative comparison, the growth stability and carbon-rich biomass observed at pilot scale in this study are consistent with trends reported for E. gracilis cultivated in real wastewater environments (Butzke et al. 2024; Barsanti et al. 2020). These findings indicate that pilot-scale assessments are essential for capturing realistic growth and carbon incorporation dynamics, which cannot be fully represented under controlled laboratory conditions. The superior performance observed in cultures with 100% effluent is consistent with the findings of He et al. (2025), who demonstrated that optimized microalgal systems for carbon capture can achieve high fixation rates when cultivated in carbon- and nutrient-rich matrices such as domestic wastewater. These authors emphasized that the use of effluents not only reduces the need for external CO₂ supplementation but also mitigates emissions associated with conventional treatment processes. Similarly, Wang et al. (2024) reported significant carbon capture capacity by microalgae cultivated in urban effluents, coupled with the simultaneous removal of nutrients and organic compounds. The mixotrophic contribution was also highlighted by Li et al. (2023a) and Viswanaathan et al. (2022), who emphasized the importance of balancing inorganic and organic carbon sources to maximize cell accumulation and photosynthetic efficiency. On the other hand, in effluent dilutions ranging from 10% to 40%, total carbon values were lower and more variable, suggesting the importance of organic carbon availability in biomass production, as it directly influences carbon assimilation and metabolic performance (Rubiyatno et al. 2021). The consistency between carbon capture results obtained at laboratory and pilot scales (bag systems) demonstrates that the scaled-up cultivation conditions maintained favorable relationships of light distribution, mixing, and gas transfer for carbon assimilation, comparable to those used at the bench scale. This observation is relevant since efficiency losses are typically expected during the transition from small to large scales, mainly due to light gradients and CO₂ diffusion limitations (Li et al. 2023a). From an environmental perspective, the use of effluents as a cultivation medium represents an integrated strategy for CO₂ emission mitigation and nutrient recovery (Wang et al. 2024; Viswanaathan et al. 2022), contributing to the carbon neutrality of the processes that generated the effluent (He et al. 2025). In this case, the beneficiary of carbon capture through the use of effluent as a medium for E. gracilis cultivation is the university’s wastewater treatment plant (WWTP). For future studies, life cycle assessments may demonstrate that the valorization of biomass produced in such systems—particularly when applied as agricultural biofertilizer or as a source of biofuels—can yield a positive environmental balance, with emissions compensated by biogenic carbon (Li et al. 2023a). 3.5 Operational aspects and challenges at pilot and large scale Despite the promising performance observed at pilot scale, the cultivation of E. gracilis in urban wastewater systems involves operational aspects that require careful consideration as cultivation volume increases. Studies investigating E. gracilis in wastewater environments have indicated that system performance may be influenced by environmental variability, particularly fluctuations in temperature and light availability, which are inherent to pilot-scale operation under realistic conditions (Barsanti et al. 2020; Nezbrytska et al. 2022). Such variability is commonly recognized as a factor that can affect growth stability, biomass productivity, and carbon assimilation in outdoor or semi-controlled cultivation systems. As cultivation systems are further scaled up, additional challenges related to light distribution, aeration efficiency, and hydrodynamic mixing become increasingly relevant. These aspects have been broadly discussed in studies involving E. gracilis cultivated in real wastewater streams, which emphasize the need for adequate system design to minimize spatial heterogeneity and maintain stable cultivation conditions (Butzke et al. 2024). Moreover, pilot-scale studies have highlighted that limitations associated with biological replication, long-term operational stability, and integration with existing wastewater treatment infrastructure remain important considerations for advancing E. gracilis -based processes toward larger-scale implementation (Barsanti et al. 2020; Nezbrytska et al. 2022). From a broader perspective, the scale-up of microalgal- and euglenoid-based wastewater treatment systems is commonly associated with increased energy demand for aeration and mixing, as well as with challenges related to light distribution, biomass harvesting, and nutrient management. These aspects have been widely recognized in recent studies as key factors influencing the technical, environmental, and economic performance of large-scale algal cultivation systems integrated with wastewater treatment. In this context, recent reviews emphasize that long-term operational stability, energy efficiency, and effective integration with existing wastewater treatment infrastructure remain critical challenges to be addressed before full-scale implementation (Cheirsilp et al. 2023; González-Camejo et al. 2021; Nagarajan et al. 2020). 4. Conclusions This study demonstrated that E. gracilis can be successfully cultivated in real urban wastewater, exhibiting high adaptability and stable growth at both laboratory and pilot scales. The comparable growth kinetics observed between scales indicate that the cultivation system can be effectively scaled up without significant losses in performance, even under non-controlled temperature conditions. The cultivation of E. gracilis in urban wastewater resulted in high removal efficiencies of chemical oxygen demand and ammoniacal nitrogen, confirming its potential for reducing organic load and nitrogenous compounds in wastewater streams. These results highlight the relevance of this microorganism as a biological component in integrated wastewater treatment strategies, particularly for organic matter and ammoniacal nitrogen removal. Urban wastewater also promoted enhanced carbon assimilation, with carbon content in the biomass exceeding 43% under undiluted effluent conditions. This indicates a strong capacity for carbon capture under mixotrophic cultivation, contributing to the mitigation of carbon emissions associated with wastewater treatment processes. The biochemical composition of the biomass was strongly influenced by the culture medium. Cultivation in mineral nutrient medium favored protein-rich biomass, whereas undiluted wastewater induced a metabolic shift toward carbohydrate accumulation, reaching values above 40%. This metabolic flexibility enables the targeted production of biomass with different biotechnological applications, depending on cultivation objectives. Although net removal of total phosphorus was not achieved under the experimental conditions due to nutrient carry-over and pH adjustment requirements, the results provide a robust proof-of-concept for integrating E. gracilis cultivation into urban wastewater treatment systems. Future studies should focus on optimizing nutrient management, inoculum preparation, and operational parameters to enhance overall nutrient removal and process efficiency. Overall, this work demonstrates the feasibility of pilot-scale E. gracilis cultivation in urban wastewater, combining organic matter reduction, carbon capture, and biomass valorization, and supports its potential role in sustainable and integrated wastewater treatment frameworks. Nevertheless, the large-scale implementation of microalgal-based technologies still requires careful consideration of operational stability, energy demand, and system integration with existing wastewater treatment infrastructure. Addressing these challenges through extended pilot operation and process optimization will be essential for advancing such systems toward full-scale application. Declarations Funding Declaration Carlos Alexandre Lutterbeck acknowledges the Brazilian “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq) for their financial support (Grant N. 440111/2022-6) and the Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (Grant Ns. 300079/2023-0 and 23/2551-0000777-0). Rosana de Cássia de Souza Schneider reports the financial support provided by the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq) for their financial support (Grant N. 306216/2022-1). Author Contribution Betina Mariela Barreto: Conceptualization, Formal analysis, Investigation, Methodology, Validation, Writing – original draftMaurício Kersting: Formal Analysis, Investigation, MethodologyCamila Rafaela Rathke: Investigation, Formal AnalysisCarlos Eduardo Flores dos Santos: Formal Analysis, Investigation, MethodologyFrancielle Pasqualotti Meinhardt: Investigation, MethodologyCamille Vitoria da Rosa: Investigation, MethodologyGabriela Bertol: Investigation, MethodologyPatrik Gustavo Wiesel: Investigation, Methodology, Writing – original draftGiséle Alves: Investigation, MethodologyRosana de Cassia de Souza Schneider: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing Carlos Alexandre Lutterbeck: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing Data Availability Data will be provided by request References Abdelfattah A, Ali SS, Ramadan H, El-Aswar EI, Eltawab R, Ho SH, Elsamahy T, Li S, El-Sheekh MM, Schagerl M, Kornaros M, Sun J (2023) Microalgae-based wastewater treatment: Mechanisms, challenges, recent advances, and future prospects. 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Environ Pollut 349:123864. doi: https://doi.org/10.1016/j.envpol.2024.123864 Razzak SA, Ali SAM, Hossain MM, deLasa H (2017) Biological CO2 fixation with production of microalgae in wastewater – A review. Renew Sustain Energy Rev 76:379–390. doi: https://doi.org/10.1016/j.rser.2017.02.038 Rubiyatno, Matsui T, Mori K, Toyama T (2021) Paramylon production by Euglena gracilis via mixotrophic cultivation using sewage effluent and waste organic compounds. Bioresour Technol Rep 15:100735. doi: https://doi.org/10.1016/j.biteb.2021.100735 Saccardo A, Bezzo F, Sforza E (2022) Microalgae growth in ultra-thin steady-state continuous photobioreactors: assessing self-shading effects. Front Bioeng Biotechnol Volume 10–2022. doi: https://doi.org/10.3389/fbioe.2022.977429 Sadigov R (2022) Rapid Growth of the World Population and Its Socioeconomic Results. The Scientific World Journal 2022 (1):8110229. doi: https://doi.org/10.1155/2022/8110229 Sayar M, Eryalçın K (2023) Investigation of Growth Performance, Proximate and Fatty Acid Composition of Freshwater (Euglena gracilis, Chlorella vulgaris) and Marine (Pavlova lutheri, Diacronema vlkanium) Microalgae. Aquat Sci Eng 0 (0):0–0. doi: https://doi.org/10.26650/ase20241303511 Sheng ALK, Bilad MR, Osman NB, Arahman N (2017) Sequencing batch membrane photobioreactor for real secondary effluent polishing using native microalgae: Process performance and full-scale projection. J Clean Prod 168:708–715. doi: https://doi.org/10.1016/j.jclepro.2017.09.083 Silveira EO, Lutterbeck CA, Machado ÊL, Rodrigues LR, Rieger A, Beckenkamp F, Lobo EA (2020) Biomonitoring of urban wastewaters treated by an integrated system combining microalgae and constructed wetlands. Sci Total Environ 705:135864. doi: https://doi.org/10.1016/j.scitotenv.2019.135864 Sluiter JB, Ruiz RO, Scarlata CJ, Sluiter AD, Templeton DW (2010) Compositional analysis of lignocellulosic feedstocks. 1. Review and description of methods. J Agric Food Chem 58 (16):9043–9053. doi: https://doi.org/10.1021/jf1008023 Sutherland DL, Ralph PJ (2020) 15 years of research on wastewater treatment high rate algal ponds in New Zealand: discoveries and future directions. N Z J Bot 58 (4):334–357. doi: https://doi.org/10.1080/0028825X.2020.1756860 Templeton DW, Laurens LML (2015) Nitrogen-to-protein conversion factors revisited for applications of microalgal biomass conversion to food, feed and fuel. Algal Res 11:359–367. doi: https://doi.org/10.1016/j.algal.2015.07.013 Tossavainen M, Lahti K, Edelmann M, Eskola R, Lampi A-M, Piironen V, Korvonen P, Ojala A, Romantschuk M (2019) Integrated utilization of microalgae cultured in aquaculture wastewater: wastewater treatment and production of valuable fatty acids and tocopherols. J Appl Phycol 31 (3):1753–1763. doi: https://doi.org/10.1007/s10811-018-1689-6 Toyama T, Kasuya M, Hanaoka T, Kobayashi N, Tanaka Y, Inoue D, Sei K, Morikawa M, Mori K (2018) Growth promotion of three microalgae, Chlamydomonas reinhardtii, Chlorella vulgaris and Euglena gracilis, by in situ indigenous bacteria in wastewater effluent. Biotechnol Biofuels 11 (1):176. doi: https://doi.org/10.1186/s13068-018-1174-0 Viswanaathan S, Perumal PK, Sundaram S (2022) Integrated Approach for Carbon Sequestration and Wastewater Treatment Using Algal–Bacterial Consortia: Opportunities and Challenges. Sustainability 14 (3):1075. doi: https://doi.org/10.3390/su14031075 Vo PHN, Kuzhiumparambil U, Kim M, Hinkley C, Pernice M, Nghiem LD, Ralph PJ (2024) Biomining using microalgae to recover rare earth elements (REEs) from bauxite. Bioresour Technol 406:131077. doi: https://doi.org/10.1016/j.biortech.2024.131077 Wang X, Hong Y, Wang Z, Yuan Y, Sun D (2024) High capacities of carbon capture and photosynthesis of a novel organic carbon-fixing microalgae in municipal wastewater: From mutagenesis, screening, ability evaluation to mechanism analysis. Water Res 257:121722. doi: https://doi.org/10.1016/j.watres.2024.121722 Wang Y, Seppanen-Laakso T, Rischer H, Wiebe MG (2018) Euglena gracilis growth and cell composition under different temperature, light and trophic conditions. PLoS One 13 (4):e0195329. doi: https://doi.org/10.1371/journal.pone.0195329 Wu M, Du M, Wu G, Lu F, Li J, Lei A, Zhu H, Hu Z, Wang J (2021) Water reuse and growth inhibition mechanisms for cultivation of microalga Euglena gracilis. Biotechnol Biofuels 14 (1):132. doi: https://doi.org/10.1186/s13068-021-01980-4 Xie W, Li X, Xu H, Chen F, Cheng KW, Liu H, Liu B (2023) Optimization of Heterotrophic Culture Conditions for the Microalgae Euglena gracilis to Produce Proteins. Mar Drugs 21 (10). doi: https://doi.org/10.3390/md21100519 Xu J, Wang X, Sun S, Zhao Y, Hu C (2017) Effects of influent C/N ratios and treatment technologies on integral biogas upgrading and pollutants removal from synthetic domestic sewage. Sci Rep 7 (1):10897. doi: https://doi.org/10.1038/s41598-017-11207-y Yaakob MA, Mohamed R, Al-Gheethi A, Aswathnarayana Gokare R, Ambati RR (2021) Influence of Nitrogen and Phosphorus on Microalgal Growth, Biomass, Lipid, and Fatty Acid Production: An Overview. Cells 10 (2). doi: https://doi.org/10.3390/cells10020393 Zhu L, Liu M, Wang Y, Zhu Z, Zhao X (2024) Euglena gracilis Protein: Effects of Different Acidic and Alkaline Environments on Structural Characteristics and Functional Properties. Foods 13 (13):2050. doi: https://doi.org/10.3390/foods13132050 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8949330","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602353035,"identity":"0bb0044b-aa3e-4b63-8919-3781b7fa78a3","order_by":0,"name":"Betina Mariela Barreto","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Betina","middleName":"Mariela","lastName":"Barreto","suffix":""},{"id":602353036,"identity":"5a287ad5-fd07-42ab-9115-9020cec2edf9","order_by":1,"name":"Maurício Kersting","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Maurício","middleName":"","lastName":"Kersting","suffix":""},{"id":602353037,"identity":"73d39c42-0310-4a3b-b6d7-c7deb27c8608","order_by":2,"name":"Carlos Eduardo Flores Santos","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Eduardo Flores","lastName":"Santos","suffix":""},{"id":602353038,"identity":"d1d7ece8-db75-4ffd-bdf2-3efbdd974b6c","order_by":3,"name":"Francielle Pasqualotti Meinhardt","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Francielle","middleName":"Pasqualotti","lastName":"Meinhardt","suffix":""},{"id":602353039,"identity":"11a9a397-301a-452d-a662-cd25d5410e8f","order_by":4,"name":"Camille Vitória da Rosa","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Camille","middleName":"Vitória da","lastName":"Rosa","suffix":""},{"id":602353042,"identity":"6f06856c-3cbc-41d4-aea6-3f9358f88fd2","order_by":5,"name":"Camila Rafaela Rathke","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Rafaela","lastName":"Rathke","suffix":""},{"id":602353043,"identity":"9b5c4e5a-7e44-418e-b78a-b8a5a0a92df7","order_by":6,"name":"Gabriela Bertol","email":"","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"","lastName":"Bertol","suffix":""},{"id":602353045,"identity":"f75ca0ad-9a4a-4764-a8ce-44a658a23582","order_by":7,"name":"Patrik Gustavo Wiesel","email":"","orcid":"","institution":"University of Santa Cruz do 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Lutterbeck","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYHACxgNAQgbM5GFgkAMKNACZB/DqOQBRDCGNSdeS2IAQxA74288+OPDhjx0Pv0Tyswdv99ikb7jd3PzhB8OdfFxaJM6kGxyc2ZbMI9lzzNxwzrO03A13DrZJ9jA8s2zAocWAIY3hMG8DM4/B8QYzaZ4Dh3M33EhsY2ZgOGyAyxYD/mcMh3n+1PPYH2b/BtTyP93gRmLzZ7xaJIC28LAd5jFg7wHZciABqKVBGp8WiRvPGIB+Oc4jceZMmeScA8mGM4EOk+wxeIZTC39/GuODD3+q5fhnpG+TeHPATp7vRvrjDz8q7uDUgtPBpGoYBaNgFIyCUYAMAEvsW8YO4/ONAAAAAElFTkSuQmCC","orcid":"","institution":"University of Santa Cruz do Sul","correspondingAuthor":true,"prefix":"","firstName":"Carlos","middleName":"Alexandre","lastName":"Lutterbeck","suffix":""}],"badges":[],"createdAt":"2026-02-23 16:55:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8949330/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8949330/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104267070,"identity":"cbb5602a-ff66-4013-8d24-6dd71842a305","added_by":"auto","created_at":"2026-03-09 20:39:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":960841,"visible":true,"origin":"","legend":"\u003cp\u003ePilot-scale system for the production of photosynthetic microorganisms consisting of 100 L plastic bags with aeration through a bubble column.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/ab9e8946db34d96ed1dc2446.png"},{"id":104267085,"identity":"688941ba-e135-449f-b858-89b894b18497","added_by":"auto","created_at":"2026-03-09 20:39:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97352,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curve by OD and Cell Density (CellD) of \u003cem\u003eE. gracilis\u003c/em\u003e in 100% urban effluent.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/0129b270a0537cd61c602ef9.png"},{"id":104267082,"identity":"16250de1-da08-4316-8bdf-aae8293ae6cb","added_by":"auto","created_at":"2026-03-09 20:39:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":276536,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature (a) and Irradiance (b) for readings taken over three consecutive days during the cultivation of \u003cem\u003eE. gracilis\u003c/em\u003e in an indoor environment, representing the entire cultivation period.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/49249a452ffe25d404950b70.png"},{"id":104267088,"identity":"af603505-45de-420f-ab87-2e2e45cdce1a","added_by":"auto","created_at":"2026-03-09 20:39:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":167767,"visible":true,"origin":"","legend":"\u003cp\u003eInfrared spectrum of \u003cem\u003eE. gracilis\u003c/em\u003e biomass samples cultivated in different media containing urban effluent.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/5e618529a0e6f6cac74b6875.png"},{"id":104267081,"identity":"81add805-aa62-45ce-b3e9-eba7189900e3","added_by":"auto","created_at":"2026-03-09 20:39:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186252,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) obtained from the FTIR spectra of samples cultivated in different proportions of effluent and in NPK medium.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/973686bf90ca1f22da9d9ace.png"},{"id":104267072,"identity":"8d554f76-fd54-4c6b-85c9-5ab2dd7af9f5","added_by":"auto","created_at":"2026-03-09 20:39:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":237053,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of amino acids present in the biomass from the analyzed systems.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/4db193ea2ee512cfcb37844b.png"},{"id":108807048,"identity":"e66098d4-4f6e-400f-8a47-10a4907ec04b","added_by":"auto","created_at":"2026-05-08 15:30:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3396044,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8949330/v1/707f19ed-8f71-46fb-8a7b-1e2f49d293c5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated cultivation of Euglena gracilis in urban wastewater: pilot-scale evaluation of organic matter removal, carbon capture and biomass valorization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe advance of urbanization and industrialization, combined with population growth, has led to a significant increase in the generation of urban wastewater (Sadigov \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When inadequately managed, these effluents cause severe environmental impacts, such as the eutrophication of water bodies and the degradation of ecosystems (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Hait et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This scenario highlights the urgent need to develop efficient and sustainable methods for wastewater treatment, making this issue a global priority due to the necessity of mitigating environmental impacts and preserving water resources (Khan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Obaideen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated the effectiveness of physicochemical and hybrid approaches based on iron-derived materials for wastewater treatment. Falyouna et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that zero-valent iron nanoparticles deposited on titanium nanowires achieved high adsorption efficiency for the removal of ciprofloxacin from aqueous solutions, highlighting the importance of nanostructured supports. Eljamal et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) showed that the combined use of iron nanoparticles and \u003cem\u003eAloe vera\u003c/em\u003e biomass significantly enhanced biogas production and process stability during anaerobic digestion of waste sludge. In addition, Maamoun et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated effective removal of phosphorus and nitrate from aqueous solutions through statistical optimization of zero-valent iron nanoparticle synthesis. Collectively, these studies indicate that iron-based technologies can achieve high contaminant removal efficiencies, although they rely on externally supplied materials and focus primarily on pollutant removal.\u003c/p\u003e \u003cp\u003eOther emerging approaches, integrated urban wastewater treatment systems associated with the cultivation of photosynthetic microorganisms have proven to be a promising technological alternative, both for the removal of organic load and for the production of high-value biomass (Morais et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cheirsilp et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Considering the microorganisms studied, \u003cem\u003eE. gracilis\u003c/em\u003e stands out as a unicellular euglenoid which, although not belonging to the true microalgae group, exhibits similar photosynthetic metabolism and physiological behavior, being able to grow under autotrophic, heterotrophic, or mixotrophic conditions. This adaptive capacity expands its potential for applications in different types of effluents, making it an ideal candidate for the remediation of contaminated urban environments (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nezbrytska et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhotosynthetic microorganism-based systems have been increasingly explored within a biorefinery approach for wastewater treatment, as they enable simultaneous nutrient removal and biomass production. These systems are effective in reducing nitrogen and phosphorus concentrations, which are key drivers of eutrophication in aquatic environments, by assimilating dissolved nutrients during biomass growth (Butzke et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nezbrytska et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Technologies such as photobioreactors and high-rate algal ponds (HRAPs) provide favorable conditions for biomass accumulation and wastewater polishing and have demonstrated promising performance at pilot and semi-industrial scales across different climatic regions (Sutherland and Ralph \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). When integrated with physical and biological processes, including sedimentation and activated sludge, these systems can further enhance treatment efficiency while reducing operational costs (Gonz\u0026aacute;lez-Camejo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWithin this biorefinery framework, \u003cem\u003eE. gracilis\u003c/em\u003e has received particular attention due to its multifunctional role in wastewater treatment. In addition to contributing to nutrient removal, this euglenophyte is capable of biofixing carbon dioxide during growth, converting inorganic carbon into biomass enriched in compounds of high economic value, which supports greenhouse gas mitigation efforts (Molazadeh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, biomass produced from wastewater-grown \u003cem\u003eE. gracilis\u003c/em\u003e has been reported as a suitable feedstock for biofuel production, including biodiesel, biogas, and bio-oil, offering a renewable alternative to fossil energy sources (Toyama et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The use of microalgal biomass for biofuel production presents advantages such as high biomass productivity and the ability to grow in non-arable or saline environments without the need for irrigation, herbicides, or pesticides (Deviram et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. gracilis\u003c/em\u003e exhibits unique characteristics that make it particularly attractive for biotechnological applications, being an excellent source of dietary proteins, vitamins, lipids, and β-1,3-glucan (paramylon), a polysaccharide exclusive to euglenoids (Khatiwada et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Its high rate of bioproduct synthesis under different culture conditions positions it as a viable competitor to microalgae such as those of the Chlorella genus, whose products are already commercially available (Sayar and Eryal\u0026ccedil;ın \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the use of this biomass, it is noteworthy that \u003cem\u003eE. gracilis\u003c/em\u003e has traditionally been used as food in some Asian countries (Mou et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In others, it is already recognized as a novel food (Nuin Garciarena et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) or generally recognized as safe (GRAS) (Guo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, when cultivated in urban wastewater, the use of \u003cem\u003eE. gracilis\u003c/em\u003e biomass faces restrictions due to the potential presence of contaminants. In such cases, its use is more viable in non-food applications, such as agriculture, biomaterial production, and biofuels (Braga et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Okeke et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe application of \u003cem\u003eE. gracilis\u003c/em\u003e in wastewater treatment has been associated with efficient nutrient removal while enabling the production of biomass enriched in compounds such as proteins, lipids, and pigments, thereby increasing its biotechnological relevance (Butzke et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its integration into effluent treatment systems has been proposed as a sustainable strategy to mitigate the environmental impacts of wastewater discharge while supporting resource recovery (Nezbrytska et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Within this context, the use of \u003cem\u003eE. gracilis\u003c/em\u003e has been framed under a biorefinery concept, in which wastewater serves not only as a treatment target but also as a cultivation medium for generating value-added bioproducts (Khatiwada et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The incorporation of \u003cem\u003eE. gracilis\u003c/em\u003e cultivation into wastewater treatment processes may also contribute to improved process efficiency and reduced operational constraints, while enabling effluent valorization through biomass utilization (Toyama et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Barsanti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Overall, this multifunctional approach combines environmental remediation with the sustainable production of high-value bioproducts, reinforcing the importance of developing integrated and efficient technologies for water and resource management (Wu et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Molinuevo-Salces et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated the feasibility of cultivating \u003cem\u003eEuglena\u003c/em\u003e spp. in domestic wastewater, primarily at laboratory scale or under controlled conditions. At lab scale, \u003cem\u003eEuglena\u003c/em\u003e sp. has been shown to grow mixotrophically in untreated domestic sewage, achieving efficient removal of organic matter and nutrients such as nitrogen and phosphorus, while producing biomass with relevant lipid content for biorefinery applications (Mahapatra et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). More recent investigations have explored mixed cultures, showing that \u003cem\u003eE. gracilis\u003c/em\u003e combined with other microalgae, such as \u003cem\u003eSelenastrum\u003c/em\u003e sp., can enhance wastewater treatment performance. In aquaculture wastewater systems, mixed cultures of \u003cem\u003eE. gracilis\u003c/em\u003e and \u003cem\u003eSelenastrum\u003c/em\u003e achieved near-complete ammonium and phosphate removal, along with high reductions in total nitrogen and phosphorus and increased biomass and lipid productivity compared to monocultures (Tossavainen et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, the ability of \u003cem\u003eEuglena\u003c/em\u003e to adapt to real wastewater streams has been demonstrated in engineered systems, such as sequencing batch membrane photobioreactors fed with secondary effluents, where native \u003cem\u003eEuglena\u003c/em\u003e strains achieved high nutrient removal efficiencies while producing lipid-containing biomass under mixotrophic conditions (Sheng et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt pilot scale, studies involving \u003cem\u003eEuglena\u003c/em\u003e in domestic wastewater have been more limited and have largely focused on its natural occurrence within complex algal\u0026ndash;bacterial communities rather than on its targeted application. In pilot-scale attached-growth pond systems treating raw domestic wastewater, \u003cem\u003eEuglena\u003c/em\u003e was identified as one of the dominant algal groups contributing to organic matter and nutrient removal (Phyu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, long-term pilot-scale multimodal algal\u0026ndash;bacterial processes treating municipal wastewater have consistently detected members of the Euglenophyceae, including \u003cem\u003eEuglena\u003c/em\u003e spp., particularly under mixotrophic conditions, where they contributed to system resilience and nutrient assimilation despite not being the dominant taxa (Mahapatra and Murthy \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although these studies highlight the ecological relevance and adaptability of \u003cem\u003eEuglena\u003c/em\u003e in pilot-scale wastewater treatment systems, they also reveal a clear gap in the literature: systematic investigations involving the deliberate inoculation of \u003cem\u003eE. gracilis\u003c/em\u003e into raw domestic wastewater at pilot scale, coupled with an integrated evaluation of growth kinetics, treatment performance, carbon assimilation, and biomass composition, remain scarce. This gap contrasts with the recognized biotechnological potential of \u003cem\u003eE. gracilis\u003c/em\u003e within biorefinery-oriented wastewater remediation frameworks (Khatiwada et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and directly motivates the need for the present pilot-scale study.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to assess the potential of \u003cem\u003eE. gracili\u003c/em\u003es for the treatment of urban wastewater, evaluating its feasibility for biomass production with biotechnological applications. In this context, it represents an innovative approach by employing \u003cem\u003eE. gracilis\u003c/em\u003e in urban effluent treatment, simultaneously exploring its capacity for nutrient removal, carbon biofixation, and the generation of biomass with biotechnological value. The use of this microorganism in wastewater represents a significant advancement over conventional systems by transforming an environmental liability into a source of renewable resources. Hence, it reinforces the importance of integrating environmental biotechnology with the principles of the circular economy, highlighting promising pathways for the development of clean and sustainable process technologies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cultivation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. gracilis\u003c/em\u003e, registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under number A863DC8, was used in this study The urban effluent employed was collected from the Wastewater Treatment Plant (WWTP) of the University of Santa Cruz do Sul (UNISC). After sampling, the effluent stood for 10 minutes to allow the sedimentation of suspended solids, and the pH was adjusted using a 5% phosphoric acid (H₃PO₄) solution, considering that \u003cem\u003eE. gracilis\u003c/em\u003e exhibits optimal growth under acidic conditions (Xie et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This adjustment also helped to minimize contamination by other microorganisms, favoring the predominant development of the target species.\u003c/p\u003e \u003cp\u003eAs a reference medium, an NPK (12:11:18) solution at a concentration of 3 g L⁻\u0026sup1; was used, which had been previously employed in the maintenance cultures of the strain and served both for inoculum production and as the control condition in the experiments (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLaboratory-scale cultures were carried out in photobioreactors with a working volume of 4 L, inoculated with \u003cem\u003eE. gracilis\u003c/em\u003e at 20% of the total volume. The system was maintained for up to three weeks under a photoperiod of 12 hours of light and 12 hours of darkness, with artificial illumination provided by 20.5 W tubular LED lamps (average of 148 \u0026micro;mol photons m⁻\u0026sup2; s⁻\u0026sup1;), in an environment without temperature control. Aeration was provided through a bubble column generated by an air compressor (VigoAr 200 Plus), using a porous stone to ensure homogeneous air distribution.\u003c/p\u003e \u003cp\u003eDifferent proportions of real raw urban wastewater were evaluated, corresponding to 10, 20, 30, 40, and 100% (v/v) of the culture medium. Considering the use of 20% inoculum and the addition of effluent percentages, the final volume was completed with NPK solution (3 g L⁻\u0026sup1;) according to the effluent proportion tested in each experimental condition. At the pilot scale, cultivation was conducted using 100% raw urban wastewater in 100 L plastic bags equipped with a bubble column, maintained indoors under artificial illumination and a 12:12 photoperiod, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe cultivation system consisted of plastic-bag photobioreactors made of low-density polyethylene (LDPE) smooth plastic bags (220 \u0026times; 40 cm; Crudo Plast), arranged vertically and illuminated by LED panels that uniformly illuminated the entire set of bags.\u003c/p\u003e \u003cp\u003eAeration was provided by a high-flow air compressor (Air 140 compressor for ornamental ponds, 220 V, 140 L min⁻\u0026sup1;), generating a continuous bubble column, with airflow limited by the size of the air inlet orifice (1 cm) into the bag. Temperature and irradiance were recorded using a HOBO data logger (UA-002-64, Onset Computer Corporation, USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Monitoring of Cell Growth\u003c/h2\u003e \u003cp\u003eThe development of the cultures was monitored weekly through cell counting in a hemocytometer using a microscope and spectrophotometric analyses performed with a UV\u0026ndash;Vis spectrophotometer, with readings at 680 nm \u0026mdash; the wavelength corresponding to the maximum absorption of chlorophyll \u0026mdash; used as an indicative parameter of cell growth. The equipment was calibrated using distilled water as a blank. In parallel, the pH of the samples was measured with a benchtop digital pH meter, allowing the monitoring of variations in this parameter throughout the experiment. At each monitoring interval, samples were collected to determine biomass, which was separated by centrifugation and subsequently dried to constant weight.\u003c/p\u003e \u003cp\u003eThe calculation of cells per mL was performed based on Eq.\u0026nbsp;1, using counts obtained with a Sedgewick\u0026ndash;Rafter counting chamber, where \u003cb\u003eN\u003c/b\u003e is the number of cells counted, \u003cb\u003eA\u003c/b\u003e is the field area in mm\u0026sup2; (1), \u003cb\u003eD\u003c/b\u003e is the chamber depth in mm (1), and \u003cb\u003eF\u003c/b\u003e is the number of fields counted (10). For the calculation of kinetic data referring to the specific growth rate (\u0026micro;), the generation rate (k), and the doubling time (td), Equations 2 to 4 were used, where x₁ and x₂ are the initial and final cell counts (cells mL⁻\u0026sup1;) at times t₁ and t₂ (days).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(CellD\\left(cell{mL}^{-1}\\right)=\\frac{Nx1000{mm}^{3}}{AxDxF}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;1\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\mu=\\frac{lnx2-lnx1}{t2-t1}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;2\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(k=\\frac{\\mu}{ln2}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;3\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({t}_{d}=\\frac{ln2}{\\mu}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;4\u003c/p\u003e \u003cp\u003eCulture monitoring was also carried out through physicochemical parameters, including Dissolved Oxygen (DO), Electrical Conductivity (EC), and Total Suspended Solids (TSS), measured using a multiparameter meter (AK-88, Akso, Italy); turbidity, measured with a digital turbidimeter (AK410 \u0026ndash; Turbidez Max, Akso, Italy).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Effluent Parameter Testing\u003c/h2\u003e \u003cp\u003eSamples were previously centrifuged and filtered through 0.45 \u0026micro;m membranes to remove suspended particles and subsequently diluted in ultrapure water to ensure the accuracy of chemical component measurements. The methods described for Chemical Oxygen Demand (COD), SMEWW 5220 B, and Ammoniacal Nitrogen (N\u0026ndash;NH₃), SMEWW 4500\u0026ndash;NH₃ B, were employed according to Standard methods for the examination of water and wastewater (APHA \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe parameters of Total Organic Carbon (TOC), Total Inorganic Carbon (TIC), and Total Nitrogen (TN) were determined using a TOC/TN elemental analyzer (TOC-LCPH/CPN, Shimadzu, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Biomass Characterization\u003c/h2\u003e \u003cp\u003eFinal biomass separation was performed by centrifugation using a refrigerated benchtop centrifuge operating at 3200 rpm for 5 minutes. After this procedure, the final supernatant was collected and reserved for physicochemical analyses, while the biomass pellet was subjected to lyophilization in a benchtop freeze dryer (LS3000, Terroni, Brazil) for preservation and subsequent characterization.\u003c/p\u003e \u003cp\u003eThe dried biomass was evaluated for gravimetric yield and biochemical composition, focusing on biomolecules of interest, including lipids, proteins, and carbohydrates. Lipid content was determined using the method adapted from Bligh and Dyer (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1959\u003c/span\u003e), as recently applied by Butzke et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Protein content was estimated from nitrogen content determined by elemental analysis (N \u0026times; 4.78), while the fixed carbon content corresponded to the percentage of total carbon measured in the biomass (Martini et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Templeton and Laurens \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Carbohydrate quantification followed the protocol described by Sluiter et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), using the concentrated acid hydrolysis method followed by analysis by high-performance liquid chromatography with refractive index detection (HPLC/RID, 20A, Shimadzu, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eResults were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, based on descriptive data analysis. Statistical analysis was performed using GraphPad Prism software (version 10.5.0). Data were initially tested for normality and homogeneity of variances to assess the suitability of parametric analyses. When these assumptions were met, differences among cultivation conditions were evaluated using one-way analysis of variance (ANOVA), followed by appropriate post hoc tests, with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistically significant differences between means are indicated by different letters.\u003c/p\u003e \u003cp\u003eWhen normality assumptions were not met, data were analyzed using the non-parametric Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s post hoc test. In cases where graphical complexity or limited replication precluded the inclusion of statistical lettering in figures or tables, statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) are reported directly in the text.\u003c/p\u003e \u003cp\u003eFor infrared spectral analysis of the biomass, the ChemoStat 4 software was used. Principal component analysis (PCA) was applied to the mean of the normalized spectra, with data preprocessed by mean-centering.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e3.1 \u003cem\u003eE. gracilis\u003c/em\u003e Growth\u003c/h2\u003e\n \u003cp\u003eAs shown in Table 1, all conditions with the addition of urban effluent exhibited cellular growth. After 21 days, the following order of cell accumulation was observed: 40% \u0026gt; 30% \u0026asymp; 20% \u0026gt; 10%. During the first week of cultivation, the greatest increase in biomass was recorded, particularly in the condition with 100% effluent addition, indicating higher nutrient bioavailability at the beginning of cultivation. This behavior is in line with that described by Liao et al. (2024), who reported that higher nutrient loads during the initial stages favor nitrogen and phosphorus assimilation by microalgae, promoting more intense exponential growth rates until the gradual depletion of available nutrients.\u003c/p\u003e\n \u003cp\u003eThe use of 100% effluent allowed for biomass production within 14 days equivalent to that obtained in 21 days under conditions with 10\u0026ndash;40% effluent, due to a faster growth kinetic (shorter cell doubling time). However, in denser or deeper cultures, factors such as self-shading, light limitation, optical interference of the effluent (color, turbidity), possible inhibitors present in the medium, and aeration constraints may attenuate this kinetic advantage and limit effective growth (Saccardo et al. 2022).\u003c/p\u003e\n \u003cp\u003eEven considering factors that may interfere with growth, among the dilutions tested, the 40% concentration showed the highest increase in cell concentration. These results highlight the feasibility of using effluent as a cultivation medium for \u003cem\u003eE. gracilis\u003c/em\u003e, as also reported by Butzke et al. (2024), who achieved similar results using swine effluent with the same \u003cem\u003eE. gracilis\u003c/em\u003e strain. However, higher concentrations of swine effluent did not yield better results, unlike what was observed with 100% urban effluent.\u003c/p\u003e\n \u003cp\u003eThe use of effluent concentrations between 10% and 40% resulted in biomass yields ranging from 0.43 to 0.69 g L⁻\u0026sup1;. The dry biomass yield varied according to the different cultivation conditions, demonstrating the adaptability of \u003cem\u003eE. gracilis\u003c/em\u003e to urban effluent. In the 100% effluent condition, the biomass obtained was closer to the value achieved using NPK as the nutrient source. However, in the pilot-scale system, harvesting must occur earlier\u0026mdash;or the culture medium must be partially renewed\u0026mdash;since after 11 days the biomass density had already entered a decline phase.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eValues of \u0026micro; in day⁻\u0026sup1;, td in days, k in divisions per day for \u003cem\u003eE. gracilis\u003c/em\u003e in medium with effluent compared to NPK.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScale\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWastewater content\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026micro; (d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaximum cultive days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003et\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ek (d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBiomass\u003c/p\u003e\n \u003cp\u003e(g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eBench\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePilot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e* The data were calculated considering experiments conducted during the same weeks; therefore, they were not performed in triplicate.\u003c/p\u003e\n \u003cp\u003eThe growth of \u003cem\u003eE. gracilis\u003c/em\u003e in raw effluent was monitored through cell density (CellD) and optical density, as shown in Fig.\u0026nbsp;2, for the culture in 100% effluent. The results indicate that \u003cem\u003eE. gracilis\u003c/em\u003e was able to assimilate the available nutrients without the need for dilution, demonstrating its potential for the bioremediation of wastewater. Monitoring carried out with the multiparameter probe also revealed significant changes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in medium quality: color, measured by absorbance at 660 nm, increased from 0.229\u0026thinsp;\u0026plusmn;\u0026thinsp;0.105 to 0.904\u0026thinsp;\u0026plusmn;\u0026thinsp;0.099, following the rise in biomass density; EC decreased from 699.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 to 162.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mS; TSS decreased from 350.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 to 81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2; and DO increased from 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 to 6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mg L⁻\u0026sup1;.\u003c/p\u003e\n \u003cp\u003eAt the pilot scale, the kinetic parameters indicated behavior similar to that observed at the laboratory scale, though with some variations associated with environmental and operational conditions. During the exponential phase, cultures in 100% effluent and in the NPK medium exhibited specific growth rates (\u0026micro;) of 0.156 and 0.118 day⁻\u0026sup1;, respectively\u0026mdash;values comparable to those obtained at the laboratory scale (0.108 and 0.162 day⁻\u0026sup1;). The observed differences can be attributed to factors such as air bubble distribution, temperature, light incidence, and biomass accumulation at the bottom of the bags, which directly influence the photosynthetic efficiency of \u003cem\u003eE. gracilis\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThe cell doubling time (td) remained within a similar range between the two scales, being 6.12 days in 100% effluent and 4.53 days in NPK medium, compared to 6.39 and 4.29 days at the laboratory scale, respectively. These results suggest that the pilot system was able to maintain yield comparable to bench scale, even without strict temperature control, as shown by the temperature and irradiance data in Fig.\u0026nbsp;3. The cell division coefficient (k) also showed little variation between scales, indicating that the adaptation of \u003cem\u003eE. gracilis\u003c/em\u003e to the larger-volume system was similar.\u003c/p\u003e\n \u003cp\u003eFigure 3 presents the monitoring of irradiance and temperature throughout the cultivation in the pilot system. Temperature (Fig.\u0026nbsp;3a) ranged from 24 to 35\u0026deg;C during the experiment, which was conducted between the spring and summer of 2024\u0026ndash;2025, a period characterized by high temperatures in southern Brazil. Although the experiments were carried out indoors, the space was not climate-controlled, resulting in significant thermal fluctuations. \u003cem\u003eE. gracilis\u003c/em\u003e was able to tolerate this gradient; however, previous studies, including those conducted in our laboratory, indicate that its growth is more favorable under controlled conditions at 25\u0026deg;C. In this context, the lack of temperature control may have acted as a limiting factor in the results obtained. For instance, Wang et al. (2018) observed that \u003cem\u003eE. gracili\u003c/em\u003es reaches its peak specific growth rate between 27 and 30\u0026deg;C, indicating that significant deviations from this range may reduce its cell multiplication efficiency.\u003c/p\u003e\n \u003cp\u003eThe recorded irradiance (Fig.\u0026nbsp;3b) varied from 28 to 40 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;, a range similar to that used by Butzke et al. (2024) during the light period of a 12:12 photoperiod. These values are also consistent with the light intensities recently reported as typical for microalgae production by Vo et al. (2024) and Adetunji et al. (2025).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e3.2 Nutrient Removal in the Cultivation of \u003cem\u003eE. gracilis\u003c/em\u003e in Urban Effluent\u003c/h2\u003e\n \u003cp\u003eThe results of the carbon, nitrogen, and phosphorus analysis in the liquid medium after cell separation are presented in Table\u0026nbsp;2. The 25.9% reduction in TOC indicates the effective removal of organic compounds present in the effluent, reflecting the ability of \u003cem\u003eE. gracilis\u003c/em\u003e to assimilate and oxidize organic matter during cultivation. Removal values of this magnitude or higher are typically observed in mixotrophic systems using photosynthetic microorganisms cultivated in urban effluents, especially when good adaptation to organic carbon sources is achieved (Mahapatra et al. 2013; Farjallah et al. 2024). In addition, the synergistic action of heterotrophic bacteria that may be present in the effluent can degrade organic compounds and contribute to TOC removal (Phyu et al. 2024). However, the efficiency of the removal process can be influenced by factors such as light availability, the presence of essential nutrients, and the carbon-to-nitrogen ratio in the medium (Pang et al. 2019). Indeed, Dang et al. (2022) and Xu et al. (2017) demonstrated that inadequate C/N ratios (either too low or too high) result in lower nitrogen and phosphorus removal efficiency, as well as negatively affecting microalgal growth rates. In mixotrophic cultures, Khanra et al. (2020) observed that adjustments in the C/N ratio modulate the assimilation of organic carbon and the protein metabolism of the cells. Similarly, Pereira et al. (2024) reported that variations in the C/N ratio influence the removal of dissolved organic carbon by microalgae. According to Xie et al. (2023), the optimal C/N ratio was 10, and in our system, whose initial C/N ratio was 1.9, it is likely that this value was outside the optimal range for \u003cem\u003eE. gracilis\u003c/em\u003e, which may have limited additional TOC removal and the utilization of residual nitrogen. These results indicate that, for the efficient use of this strain in treatment systems, future studies should evaluate the influence of different nutrient proportions on the growth and cellular metabolism of this species.\u003c/p\u003e\n \u003cp\u003eThe expressive 99.1% removal of inorganic carbon (IC) indicates the fixation of inorganic carbon by \u003cem\u003eE. gracilis\u003c/em\u003e. This result can be explained by the fact that dissolved inorganic carbon represents the main carbon source for photosynthesis in microalgae (Kusi et al. 2024). Additionally, this reduction may be related to the cultivation of \u003cem\u003eE. gracilis\u003c/em\u003e as an efficient carbon fixation system. Razzak et al. (2017) highlighted that photobiological systems can remove more than 80% of inorganic carbon from effluents, depending on cultivation conditions such as aeration and light availability. The 69.5% removal of total carbon (TC) reflects the combined contribution of both organic and inorganic carbon assimilation processes.\u003c/p\u003e\n \u003cp\u003eSimilar results have been reported in studies that employed microalgae for wastewater treatment, where TC removal ranged from 50% to 80%, depending on effluent composition and experimental conditions (Lu 2025). However, Toyama et al. (2018) noted that the efficiency of TC removal is affected by the presence of symbiotic bacteria, which assist in the conversion of organic matter and promote the release of CO₂ for fixation into the biomass.\u003c/p\u003e\n \u003cp\u003eThe increase observed in total nitrogen and total phosphorus concentrations at the end of treatment is related to the addition of the \u003cem\u003eE. gracilis\u003c/em\u003e inoculum previously cultivated in synthetic medium containing NPK, which introduced residual nutrients into the system. This effect, however, was less pronounced when an inoculum previously adapted to the effluent was used, indicating that prior adaptation constitutes an effective strategy to minimize external nutrient input during the treatment process. Nevertheless, considering that the culture was initiated with an isolated \u003cem\u003eEuglena\u003c/em\u003e strain, supplementation with NPK in the inoculum and pH adjustment were necessary to ensure its predominance in the microbial consortium. In applied systems, longer hydraulic retention times and integration with other nature-based technologies (such as constructed wetlands or high-rate algal ponds) may enhance nutrient removal and reduce the dependence on external input addition (Sutherland and Ralph 2020). It is understood that microalgae can represent one component of a broader treatment system, as they remove emerging contaminants and nutrients, increase dissolved oxygen, reduce chemical oxygen demand, and establish a symbiotic environment with bacteria during the treatment process (Nguyen et al. 2022; Bang Truong et al. 2024; Abdelfattah et al. 2023).\u003c/p\u003e\n \u003cp\u003eThe evaluation of treatment efficiency applied to the effluent generated at the university campus demonstrated that the system employing \u003cem\u003eE. gracilis\u003c/em\u003e met some of the emission standards established by CONSEMA Resolution No. 355/2017, particularly concerning Chemical Oxygen Demand (COD) and Ammoniacal Nitrogen (N\u0026ndash;NH₃).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eParameters of the effluent before and after treatment with \u003cem\u003eE. gracili\u003c/em\u003es in 100% effluent.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInitial\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 3 weeks\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRemoval (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eInorganic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNitrogen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.31\u0026thinsp;\u0026plusmn;\u0026thinsp;17.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmmoniacal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhosphorus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eEletrical Conductivity (\u0026micro;Scm⁻\u0026sup1;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e793\u0026thinsp;\u0026plusmn;\u0026thinsp;179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eChemical Oxygen Demand (COD) (mg L⁻\u0026sup1;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e˂2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eTurbidity (NTU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Suspended Solids (TSS)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e350.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e*pH adjusted for cultivation\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eCOD removal reached 98%, a result superior to that obtained in the study conducted by Butzke et al. (2024), who reported a reduction efficiency of 44% in diluted swine wastewater (25%). This difference may be attributed to the higher nutrient concentration in that type of effluent, which initially presented COD values of 3101 mg L⁻\u0026sup1;\u0026mdash;substantially higher than the initial values of the raw urban effluent used in the present study. Kim et al. (2021) demonstrated that the cultivation of \u003cem\u003eE. gracilis\u003c/em\u003e in tomato processing wastewater under continuous illumination (24 h light) achieved a COD removal of 23% at pH 3. Comparison with these studies indicates that the environmental conditions adopted in the present work\u0026mdash;such as the 12:12 light\u0026ndash;dark photoperiod and acidic pH\u0026mdash;likely favored the optimal utilization of organic carbon fractions present in the wastewater. This emphasizes the importance of environmental conditions for achieving efficient nutrient removal by photosynthetic organisms such as Euglena.\u003c/p\u003e\n \u003cp\u003eIt is also important to highlight the remarkable efficiency of the system in the removal of N\u0026ndash;NH₃, reaching values close to 97%, corresponding to a final concentration below 0.5 mg L⁻\u0026sup1;. As expected for \u003cem\u003eE. gracilis\u003c/em\u003e, ammoniacal nitrogen is primarily removed from the culture medium (He et al. 2025). This performance is comparable to that reported by Silveira et al. (2020) in an integrated system (microalgae associated with constructed wetlands), which achieved 98% nitrogen removal. Kuroda et al. (2018) also demonstrated high nutrient removal potential in primary sedimentation tank effluent, where during 14 days of cultivation \u003cem\u003eE. gracilis\u003c/em\u003e almost completely removed ammonium (NH₄⁺\u0026ndash;N), resulting in final concentrations below 0.2 mg L⁻\u0026sup1;.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.3 Biomass Yield and Composition\u003c/h2\u003e\n \u003cp\u003eThe biochemical composition of \u003cem\u003eE. gracilis\u003c/em\u003e biomass varied significantly according to the proportion of urban effluent in the culture medium (Table\u0026nbsp;4), reflecting metabolic adjustments to different nutrient availability and physicochemical conditions.\u003c/p\u003e\n \u003cp\u003eThe results indicate that the highest protein synthesis occurred under control conditions (NPK only), both at pilot scale (40.0%) and laboratory scale (49.9%). These higher values are associated with media rich in readily assimilable inorganic nitrogen, which is essential for the synthesis of structural and enzymatic proteins (He et al. 2025; Yaakob et al. 2021). The progressive addition of effluent resulted in a significant reduction in protein content, especially at the 100% effluent concentration, where the value decreased to 23.9%.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBiochemical composition of \u003cem\u003eE. gracilis\u003c/em\u003e dry biomass cultivated at different concentrations of urban effluent.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eWastewater concentration (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eBiochemical composition (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLipids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarbohydrate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ePilot-scale\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl (only NPK)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eBench-scale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl (only NPK)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eMeans followed by the same letter do not differ significantly according to analysis of variance (ANOVA) at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n \u003cp\u003eThis reduction can be attributed to the higher C/N ratio in urban effluent, characterized by a relative excess of organic carbon and a lower availability of assimilable nitrogen. According to Xie et al. (2023), E. \u003cem\u003egracilis\u003c/em\u003e tends to redirect its metabolism as the C/N ratio increases, reducing protein synthesis while favoring the formation of storage compounds. Therefore, mineral media (NPK) or effluent diluted up to 20% are more suitable when the goal is to maximize the protein content of the biomass.\u003c/p\u003e\n \u003cp\u003eAn opposite trend was observed for carbohydrates, whose content increased with the rise in effluent proportion. At the laboratory scale, values ranged from 13.8% (control) to 40.8% (100% effluent), indicating intense carbohydrate accumulation under high organic load. This increase is related to nitrogen limitation and metabolic stress, which induce \u003cem\u003eE. gracilis\u003c/em\u003e to accumulate the polysaccharide paramylon, an energy reserve analogous to starch. According to Bakku et al. (2023) and Toyama et al. (2018), this response is typical of mixotrophic or heterotrophic cultures subjected to carbon excess and inorganic nutrient restriction.\u003c/p\u003e\n \u003cp\u003eIn the pilot system, the same trend was observed, although less pronounced, suggesting that physical factors such as light dispersion, thermal gradients, and aeration limitation may modulate the biochemical response of the biomass (Farjallah et al. 2024).\u003c/p\u003e\n \u003cp\u003eThus, the presence of urban effluent significantly altered intracellular carbon allocation, redirecting the metabolism of \u003cem\u003eE. gracilis\u003c/em\u003e from active protein synthesis toward the accumulation of energy reserves. The effluent provides a complex mixture of organic and inorganic compounds that modifies nutrient availability, turbidity, and light penetration in the medium, thereby affecting photosynthetic activity (Abdelfattah et al. 2023). In addition, the possible symbiotic activity of heterotrophic bacteria present in the effluent may contribute to the mineralization of organic matter and the release of assimilable CO₂ (Toyama et al. 2018), reinforcing the mixotrophic nature of the culture.\u003c/p\u003e\n \u003cp\u003eThese findings corroborate the results reported by Alves et al. (2024) for the same strain cultivated in NPK medium supplemented with a high-organic-load residue, which exhibited low lipid (8.4%) and protein (35.9%) contents and a high carbohydrate content (66.5%).\u003c/p\u003e\n \u003cp\u003eConsidering that the same samples used in the laboratory-scale experiments were also analyzed by FTIR, a clear agreement was observed between the results obtained from the different analytical approaches. The characteristic bands of the main compound groups are highlighted in Fig.\u0026nbsp;4, showing intense signals around 1100 cm⁻\u0026sup1;, attributed to C\u0026ndash;O\u0026ndash;C and C\u0026ndash;O vibrations typical of carbohydrates (Ferro et al. 2019). Bands of lower intensity were also observed near 1650 cm⁻\u0026sup1;, corresponding to the C\u0026thinsp;=\u0026thinsp;O stretching (Amide I) of protein chains, and around 2900 cm⁻\u0026sup1;, associated with the C\u0026ndash;H vibrations of methyl and methylene groups present in lipids (Mahapatra et al. 2013; Zhu et al. 2024; Murdock and Wetzel 2009).\u003c/p\u003e\n \u003cp\u003eThe spectra were subjected to principal component analysis (PCA), as shown in Fig.\u0026nbsp;4. The PC1 axis explained 91.27% of the total variance, representing the main source of difference among the samples. This component primarily discriminated the presence of functional groups associated with carbohydrates, since the samples cultivated in medium containing 100% effluent exhibited a higher proportion of these compounds. The PC2 axis explained 5.34% of the variance and reflected variations related to protein content: samples located on the negative side of PC2 and positive side of PC1 exhibited higher protein levels and lower carbohydrate content, except for the sample cultivated in NPK medium, positioned in the positive quadrant of this component, which showed high protein content and lower lipid content. These results corroborate the infrared spectroscopic analyses, confirming through PCA of the spectra the trend observed in the data presented in Table\u0026nbsp;4.\u003c/p\u003e\n \u003cp\u003eRegarding the proteins of \u003cem\u003eE. gracilis\u003c/em\u003e obtained from the different systems tested, it was observed that they are mainly composed of the amino acids arginine, glutamic acid, aspartic acid, and alanine, indicating a protein profile with relevant amounts of essential amino acids, as shown in Fig.\u0026nbsp;5. These values were similar to those reported by Butzke et al. (2024) for \u003cem\u003eE. gracilis\u003c/em\u003e cultivated in swine wastewater for the purpose of producing a germination biostimulant. Most amino acids showed a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the cultivation conditions. Notably, arginine concentration was markedly higher in the biomass obtained from the bench-scale system cultivated with 100% urban wastewater compared to the other conditions, and this amino acid is of particular importance for plant metabolism. This composition reinforces the potential application of the biomass in agricultural bioinput formulations (Xie et al. 2023).\u003c/p\u003e\n \u003cp\u003eOn the other hand, cultures grown under conditions that favor carbohydrate accumulation\u0026mdash;particularly paramylon\u0026mdash;make the biomass a promising source of paramylon nanofibers (Azuma and Yamamoto 2024), which can be hydrolyzed into glucose and subsequently used for bioethanol production. Research has demonstrated that, after hydrolysis and fermentation processes, ethanol can be obtained from \u003cem\u003eE. gracilis\u003c/em\u003e biomass (Mou et al. 2024).\u003c/p\u003e\n \u003cp\u003eConsidering that the biomass obtained from pilot-scale cultivation in 100% effluent presented an average concentration of 0.58 g L⁻\u0026sup1; with 40% total carbohydrates, an equivalent fraction of 0.23 g L⁻\u0026sup1; of carbohydrates was estimated. Of this total, 63.9% correspond to glucose derived from paramylon hydrolysis, resulting in approximately 0.148 g L⁻\u0026sup1; of glucose available for fermentation. Based on the stoichiometry of glucose conversion to ethanol (C₆H₁₂O₆ \u0026rarr; 2 C₂H₅OH\u0026thinsp;+\u0026thinsp;2 CO₂) by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the theoretical ethanol yield is 0.51 g g⁻\u0026sup1; of glucose, corresponding to a production potential of 0.075 g L⁻\u0026sup1; of ethanol. Although this value is modest, it demonstrates that the biomass of \u003cem\u003eE. gracilis\u003c/em\u003e cultivated in effluent represents an alternative source of fermentable substrate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.4 Carbon Capture\u003c/h2\u003e\n \u003cp\u003eCarbon capture varied significantly among the evaluated conditions, demonstrating the effect of medium composition and cultivation scale on the metabolism of \u003cem\u003eE. gracilis\u003c/em\u003e (Table\u0026nbsp;4). It was observed that the use of 100% urban effluent promoted the highest carbon incorporation into the biomass, both at the laboratory scale (43.66%) and at the pilot scale (43.33%), surpassing the values obtained in the NPK controls. This behavior indicates that the effluent provided not only inorganic carbon sources but also assimilable organic fractions, which is characteristic of mixotrophic systems. Recent studies have reinforced that the presence of dissolved organic carbon in effluents stimulates carbon assimilation by microalgae, resulting in high net fixation rates and potentially carbon-negative systems due to the simultaneous utilization of atmospheric CO₂ and available organic carbon (Abdelfattah et al. 2023; He et al. 2025; Wang et al. 2024).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eElemental analysis of carbon captured in the biomass of \u003cem\u003eE. gracilis\u003c/em\u003e cultivated in urban effluent medium.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEscale\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEffluent (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarbon (%) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePilot\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLaboratory\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eCarbon capture in microalgal systems is intrinsically linked to biomass growth and carbon allocation within the cells. In this context, the pilot-scale cultivation of \u003cem\u003eE.gracilis\u003c/em\u003e in the present study showed stable growth kinetics and consistent incorporation of carbon into the biomass under realistic operational conditions. Similar behavior has been reported in previous pilot and semi-pilot studies with \u003cem\u003eE. gracilis\u003c/em\u003e, which demonstrated the capacity of this species to sustain growth and metabolic activity in wastewater-based systems subject to environmental variability (Nezbrytska et al. 2022).\u003c/p\u003e\n \u003cp\u003eAlthough methodological differences prevent direct quantitative comparison, the growth stability and carbon-rich biomass observed at pilot scale in this study are consistent with trends reported for \u003cem\u003eE. gracilis\u003c/em\u003e cultivated in real wastewater environments (Butzke et al. 2024; Barsanti et al. 2020). These findings indicate that pilot-scale assessments are essential for capturing realistic growth and carbon incorporation dynamics, which cannot be fully represented under controlled laboratory conditions.\u003c/p\u003e\n \u003cp\u003eThe superior performance observed in cultures with 100% effluent is consistent with the findings of He et al. (2025), who demonstrated that optimized microalgal systems for carbon capture can achieve high fixation rates when cultivated in carbon- and nutrient-rich matrices such as domestic wastewater. These authors emphasized that the use of effluents not only reduces the need for external CO₂ supplementation but also mitigates emissions associated with conventional treatment processes. Similarly, Wang et al. (2024) reported significant carbon capture capacity by microalgae cultivated in urban effluents, coupled with the simultaneous removal of nutrients and organic compounds. The mixotrophic contribution was also highlighted by Li et al. (2023a) and Viswanaathan et al. (2022), who emphasized the importance of balancing inorganic and organic carbon sources to maximize cell accumulation and photosynthetic efficiency.\u003c/p\u003e\n \u003cp\u003eOn the other hand, in effluent dilutions ranging from 10% to 40%, total carbon values were lower and more variable, suggesting the importance of organic carbon availability in biomass production, as it directly influences carbon assimilation and metabolic performance (Rubiyatno et al. 2021).\u003c/p\u003e\n \u003cp\u003eThe consistency between carbon capture results obtained at laboratory and pilot scales (bag systems) demonstrates that the scaled-up cultivation conditions maintained favorable relationships of light distribution, mixing, and gas transfer for carbon assimilation, comparable to those used at the bench scale. This observation is relevant since efficiency losses are typically expected during the transition from small to large scales, mainly due to light gradients and CO₂ diffusion limitations (Li et al. 2023a).\u003c/p\u003e\n \u003cp\u003eFrom an environmental perspective, the use of effluents as a cultivation medium represents an integrated strategy for CO₂ emission mitigation and nutrient recovery (Wang et al. 2024; Viswanaathan et al. 2022), contributing to the carbon neutrality of the processes that generated the effluent (He et al. 2025). In this case, the beneficiary of carbon capture through the use of effluent as a medium for \u003cem\u003eE. gracilis\u003c/em\u003e cultivation is the university\u0026rsquo;s wastewater treatment plant (WWTP). For future studies, life cycle assessments may demonstrate that the valorization of biomass produced in such systems\u0026mdash;particularly when applied as agricultural biofertilizer or as a source of biofuels\u0026mdash;can yield a positive environmental balance, with emissions compensated by biogenic carbon (Li et al. 2023a).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.5 Operational aspects and challenges at pilot and large scale\u003c/h2\u003e\n \u003cp\u003eDespite the promising performance observed at pilot scale, the cultivation of \u003cem\u003eE. gracilis\u003c/em\u003e in urban wastewater systems involves operational aspects that require careful consideration as cultivation volume increases. Studies investigating \u003cem\u003eE. gracilis\u003c/em\u003e in wastewater environments have indicated that system performance may be influenced by environmental variability, particularly fluctuations in temperature and light availability, which are inherent to pilot-scale operation under realistic conditions (Barsanti et al. 2020; Nezbrytska et al. 2022). Such variability is commonly recognized as a factor that can affect growth stability, biomass productivity, and carbon assimilation in outdoor or semi-controlled cultivation systems.\u003c/p\u003e\n \u003cp\u003eAs cultivation systems are further scaled up, additional challenges related to light distribution, aeration efficiency, and hydrodynamic mixing become increasingly relevant. These aspects have been broadly discussed in studies involving \u003cem\u003eE. gracilis\u003c/em\u003e cultivated in real wastewater streams, which emphasize the need for adequate system design to minimize spatial heterogeneity and maintain stable cultivation conditions (Butzke et al. 2024). Moreover, pilot-scale studies have highlighted that limitations associated with biological replication, long-term operational stability, and integration with existing wastewater treatment infrastructure remain important considerations for advancing \u003cem\u003eE. gracilis\u003c/em\u003e-based processes toward larger-scale implementation (Barsanti et al. 2020; Nezbrytska et al. 2022).\u003c/p\u003e\n \u003cp\u003eFrom a broader perspective, the scale-up of microalgal- and euglenoid-based wastewater treatment systems is commonly associated with increased energy demand for aeration and mixing, as well as with challenges related to light distribution, biomass harvesting, and nutrient management. These aspects have been widely recognized in recent studies as key factors influencing the technical, environmental, and economic performance of large-scale algal cultivation systems integrated with wastewater treatment. In this context, recent reviews emphasize that long-term operational stability, energy efficiency, and effective integration with existing wastewater treatment infrastructure remain critical challenges to be addressed before full-scale implementation (Cheirsilp et al. 2023; Gonz\u0026aacute;lez-Camejo et al. 2021; Nagarajan et al. 2020).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study demonstrated that \u003cem\u003eE. gracilis\u003c/em\u003e can be successfully cultivated in real urban wastewater, exhibiting high adaptability and stable growth at both laboratory and pilot scales. The comparable growth kinetics observed between scales indicate that the cultivation system can be effectively scaled up without significant losses in performance, even under non-controlled temperature conditions. The cultivation of \u003cem\u003eE. gracilis\u003c/em\u003e in urban wastewater resulted in high removal efficiencies of chemical oxygen demand and ammoniacal nitrogen, confirming its potential for reducing organic load and nitrogenous compounds in wastewater streams. These results highlight the relevance of this microorganism as a biological component in integrated wastewater treatment strategies, particularly for organic matter and ammoniacal nitrogen removal.\u003c/p\u003e \u003cp\u003eUrban wastewater also promoted enhanced carbon assimilation, with carbon content in the biomass exceeding 43% under undiluted effluent conditions. This indicates a strong capacity for carbon capture under mixotrophic cultivation, contributing to the mitigation of carbon emissions associated with wastewater treatment processes. The biochemical composition of the biomass was strongly influenced by the culture medium. Cultivation in mineral nutrient medium favored protein-rich biomass, whereas undiluted wastewater induced a metabolic shift toward carbohydrate accumulation, reaching values above 40%. This metabolic flexibility enables the targeted production of biomass with different biotechnological applications, depending on cultivation objectives.\u003c/p\u003e \u003cp\u003eAlthough net removal of total phosphorus was not achieved under the experimental conditions due to nutrient carry-over and pH adjustment requirements, the results provide a robust proof-of-concept for integrating \u003cem\u003eE. gracilis\u003c/em\u003e cultivation into urban wastewater treatment systems. Future studies should focus on optimizing nutrient management, inoculum preparation, and operational parameters to enhance overall nutrient removal and process efficiency. Overall, this work demonstrates the feasibility of pilot-scale \u003cem\u003eE. gracilis\u003c/em\u003e cultivation in urban wastewater, combining organic matter reduction, carbon capture, and biomass valorization, and supports its potential role in sustainable and integrated wastewater treatment frameworks.\u003c/p\u003e \u003cp\u003eNevertheless, the large-scale implementation of microalgal-based technologies still requires careful consideration of operational stability, energy demand, and system integration with existing wastewater treatment infrastructure. Addressing these challenges through extended pilot operation and process optimization will be essential for advancing such systems toward full-scale application.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCarlos Alexandre Lutterbeck acknowledges the Brazilian \u0026ldquo;Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico\u0026rdquo; (CNPq) for their financial support (Grant N. 440111/2022-6) and the Funda\u0026ccedil;\u0026atilde;o de Amparo a Pesquisa do Estado do Rio Grande do Sul (Grant Ns. 300079/2023-0 and 23/2551-0000777-0). Rosana de C\u0026aacute;ssia de Souza Schneider reports the financial support provided by the \u0026ldquo;Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico\u0026rdquo; (CNPq) for their financial support (Grant N. 306216/2022-1).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBetina Mariela Barreto: Conceptualization, Formal analysis, Investigation, Methodology, Validation, Writing \u0026ndash; original draftMaur\u0026iacute;cio Kersting: Formal Analysis, Investigation, MethodologyCamila Rafaela Rathke: Investigation, Formal AnalysisCarlos Eduardo Flores dos Santos: Formal Analysis, Investigation, MethodologyFrancielle Pasqualotti Meinhardt: Investigation, MethodologyCamille Vitoria da Rosa: Investigation, MethodologyGabriela Bertol: Investigation, MethodologyPatrik Gustavo Wiesel: Investigation, Methodology, Writing \u0026ndash; original draftGis\u0026eacute;le Alves: Investigation, MethodologyRosana de Cassia de Souza Schneider: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing Carlos Alexandre Lutterbeck: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be provided by request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelfattah A, Ali SS, Ramadan H, El-Aswar EI, Eltawab R, Ho SH, Elsamahy T, Li S, El-Sheekh MM, Schagerl M, Kornaros M, Sun J (2023) Microalgae-based wastewater treatment: Mechanisms, challenges, recent advances, and future prospects. 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Cells 10 (2). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells10020393\u003c/span\u003e\u003cspan address=\"10.3390/cells10020393\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu L, Liu M, Wang Y, Zhu Z, Zhao X (2024) Euglena gracilis Protein: Effects of Different Acidic and Alkaline Environments on Structural Characteristics and Functional Properties. Foods 13 (13):2050. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods13132050\u003c/span\u003e\u003cspan address=\"10.3390/foods13132050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Euglena gracilis, urban wastewater, phycoremediation, biomass valorization, circular bioeconomy, carbon capture","lastPublishedDoi":"10.21203/rs.3.rs-8949330/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8949330/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the cultivation of \u003cem\u003eEuglena gracilis\u003c/em\u003e in real urban wastewater as a strategy for organic matter removal, carbon capture, and biomass valorization. Cultivation was conducted at laboratory and pilot scales using different proportions of urban wastewater and a mineral nutrient medium as control. \u003cem\u003eE. gracilis\u003c/em\u003e successfully adapted to the wastewater from a university wastewater treatment plant, showing growth kinetics at pilot scale comparable to laboratory-scale systems. High removal efficiencies were achieved for chemical oxygen demand (up to 98.0%) and ammoniacal nitrogen (up to 91.6%), demonstrating the potential of this organism for organic load reduction.\u003c/p\u003e \u003cp\u003eCultivation in undiluted wastewater enhanced carbon incorporation into the biomass, reaching values above 43%, indicating strong carbon assimilation under mixotrophic conditions. The biochemical composition of the biomass varied according to the culture medium, with a clear metabolic shift from protein-rich biomass in mineral medium to carbohydrate accumulation (up to 40.8%) in wastewater-based cultures. These results demonstrate the feasibility of integrating \u003cem\u003eE. gracilis\u003c/em\u003e cultivation into urban wastewater systems at pilot scale, combining organic matter removal with carbon capture and the generation of biomass with biotechnological potential.\u003c/p\u003e","manuscriptTitle":"Integrated cultivation of Euglena gracilis in urban wastewater: pilot-scale evaluation of organic matter removal, carbon capture and biomass valorization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 20:39:20","doi":"10.21203/rs.3.rs-8949330/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"efa9a374-e938-4c39-9dd2-f357612161e4","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-08T03:38:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T03:28:38+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"117906910621627516463132729149653809467","date":"2026-05-04T01:38:07+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T03:54:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 20:39:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8949330","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8949330","identity":"rs-8949330","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}