Phycoremediation potential of Nostoc ellipsosporum and Spirulina subsalsa for pollutant removal from real textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass

Phycoremediation potential of Nostoc ellipsosporum and Spirulina subsalsa for pollutant removal from real textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass | 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 Phycoremediation potential of Nostoc ellipsosporum and Spirulina subsalsa for pollutant removal from real textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass Chathurika Bandara, Yuansong Wei, Charmalie Abayasekara, Renuka Ratnayake This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6947833/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present study investigated the potential of two cyanobacterial species, namely Nostoc ellipsosporum (NE) and Spirulina subsalsa (SS) to remediate textile wastewater (TWW) while obtaining wastewater-grown biomass for biodiesel production. This study reports their multi-faceted benefits using unsterilized and undiluted (100%) TWW for the first time. Both cyanobacterial species were cultivated in TWW under greenhouse conditions, focusing on their growth, TWW decolorization, pollutant removal, phytotoxicity, lipid content, and fatty acid profile. Results demonstrated significant growth and decolorization of TWW by both species, highlighting their potential for sustainable TWW treatment. NE-treated TWW (NE-TWW) achieved a chemical oxygen demand (COD) removal efficiency of 96.48%, while SS-treated TWW (SS-TWW) reached 93.11%. Ammonia removal rates were recorded at 76.28% for NE-TWW and 96.86% for SS-TWW. NE-TWW and SS-TWW achieved nitrate removal of 69% and 73%, respectively. Phosphate removal was 81.14% for NE-TWW and 33.33% for SS-TWW. Seed germination studies showed enhanced shoot and root development of green gram (Vigna radiata) when irrigated with NE-TWW and SS-TWW, suggesting potential applications for irrigation. Lipid yields of 21.5% and 25% were recorded from TWW-grown biomass of NE and SS, respectively. Both species exhibited favorable fatty acid methyl ester (biodiesel) profiles, dominated by palmitic acid (C16:0), oleic acid (C18:1), stearic acid (C18:0), lauric acid (C12:0), and myristic acid (C14:0), indicating their suitability as biodiesel feedstocks. This integrated approach not only provides an effective solution for TWW treatment but also offers a sustainable feedstock for biodiesel production and an alternative water source for irrigation, aligning with the circular bioeconomy principles. cyanobacteria textile wastewater bioremediation phytotoxicity total lipid content biodiesel Figures Figure 1 Figure 2 Figure 3 Introduction The textile industry is one of the most water-intensive industries, consuming large volumes of water for different manufacturing processes like sizing, de-sizing, scouring, bleaching, mercerizing, dyeing, and printing (Brar et al., 2019 ; Khan & Malik, 2014 ), in turn generating significant amounts of wastewater. These wastewaters are typically highly-colored due to the high load of toxic dyes, and contain heavy metals, recalcitrant organic compounds, and excessive amounts of inorganic ions. Textile wastewater (TWW) is toxic, carcinogenic and mutagenic due to the presence of synthetic dyes such as azo, anthraquinone, and phthalocyanine dyes (Khan & Malik, 2014 ; Khatri et al., 2018 ). They reduce light penetration and photosynthetic activity in water bodies, consequently limiting oxygen supply to aquatic organisms and inhibiting their growth (Kishor et al., 2020 ). The discharge of nutrient-rich (nitrogen and phosphorous) wastewater is also a major source of water pollution owing to eutrophication. Thus, nutrient removal has become crucial alongside organic pollutant removal (Hasan et al., 2021 ). Conventional wastewater treatment methods, such as chemical coagulation, electro-flocculation, adsorption, advanced oxidation processes, and membrane technology, often present challenges related to high costs, secondary pollution, sludge management and inefficiency in removing complex contaminants (Bharagava & Bhimrao, 2018 ; Mona et al., 2020 ). As a sustainable and cost-effective alternative, bioremediation of TWW using cyanobacteria has gained significant attention due to their ability to metabolize pollutants while simultaneously consuming them to produce valuable biomass. This biomass could be employed in generation of biofuels, biopolymers and biofertilizers etc (Sadvakasova et al., 2021 ). Cyanobacteria, also known as blue-green algae are prokaryotic photosynthetic microorganisms known for their rapid growth and high tolerance to extreme environmental conditions. They have demonstrated remarkable potential in the bioremediation of textile effluents (Mathimani et al., 2024 ; Selvaraj & Arivazhagan, 2024 ). As photosynthetic organisms, cyanobacteria can release O 2 while assimilating CO 2, unlike bacteria-based wastewater treatment where a significant amount of CO 2 is emitted. Cyanobacteria employ several mechanisms in TWW bioremediation (Tsolcha et al., 2017 ). These mechanisms include dye decolorization, biosorption, biomineralization, biotransformation, bioaccumulation and enzymatic biodegradation, while sequestering heavy metals, and assimilating inorganic and organic compounds, leading to improved water quality (Mona et al., 2020 ; Touliabah et al., 2022 ). Furthermore, cyanobacteria accumulate high lipid contents under nutrient-stress conditions, making them a promising feedstock for biodiesel production (M. M. El-Sheekh et al., 2023 ). Hence, integrating TWW bioremediation with successive biodiesel production not only addresses environmental concerns but also enhances the economic feasibility of the process. However, the complexity of wastewater and algal ecological characteristics induce techno-economic challenges for its industrial implementation (Brar et al., 2019 ; Nguyen et al., 2022 ). This study explored the dual role of two filamentous cyanobacterial species, namely Nostoc ellipsosporum (NE) and Spirulina subsalsa (SS) in TWW remediation and their potential for biodiesel generation. Limited studies have been conducted to explore the potential of cyanobacteria in TWW bioremediation, compared to other wastewater types (Cuellar-Bermudez et al., 2017 ; M. El-Sheekh et al., 2022 ). To the best of our knowledge, this could be the first report of utilizing above-mentioned cyanobacterial species to treat unsterilized and undiluted (100%) TWW under greenhouse conditions without addition of any extra nutrients, with a key focus of investigating the practicability of this strategy, saving energy and freshwater usage, in terms of sterilization and dilution, respectively. The study aimed to evaluate the growth performance of NE and SS in TWW, while focusing on their ability to decolorize TWW. The study assessed their effectiveness in removing COD and inorganic nutrients (ammoniacal nitrogen, nitrate, phosphate) from TWW. Moreover, this study investigated the impact of treated TWW on seed germination, particularly focusing on shoot and root development. Furthermore, the research objectives were to explore the total lipid content of TWW-grown biomass while assessing their fatty acid profile for biodiesel production. Thus, this research aimed to establish an eco-friendly and cost-effective approach to TWW treatment while employing TWW-grown biomass for biodiesel production. The findings of this study could pave the way for a circular bioeconomy, where wastewater treatment and energy generation are seamlessly integrated for environmental and industrial sustainability. Materials & Methods Cyanobacterial species and collection of textile wastewater The cyanobacterial cultures (NE and SS) were obtained from the NIFS-Sri Lanka culture collection (NIFS-SLCC), registered in the World Data Centre for Microorganisms (WDCM) (Reg. No: 1245) and maintained in BG-11 (Stanier et al., 1971) medium in 250 ml conical flasks at room temperature under 2500 lux of white fluorescent light till exponentially growing phase is attained. TWW was collected from a leading textile company in the Biyagama Export Processing Zone (6.9775° N, 79.9800° E), Sri Lanka and their initial physicochemical parameters were assessed in accordance with the established guidelines provided by American Public Health Association, (APHA, 2012). The pH, electric conductivity (EC), salinity, total dissolved solids (TDS) were measured onsite using a portable multiparameter (Oyster TM , NH, USA) (Table 1). Evaluation of cyanobacterial growth and TWW decolorization A 10% (v/v) homogenous algal suspension was inoculated into transparent glass tanks containing a total volume of 20 L of unsterilized and undiluted (100%) TWW. They were maintained under greenhouse conditions with a photoperiod of 12:12 h employing continuous air bubbling into the tanks using aerator pumps. The growth of NE and SS was monitored during three-day intervals by measuring optical density at 680 nm using an UV–visible spectrophotometer (Agilent Carry 60, CA, USA) over a period of 28 days to monitor their growth. To assess the decolorization of the TWW by NE and SS, an UV-spectral scan was done initially within the visible range (400 nm to 800 nm) to find the maximum absorbance wavelength (λ max ) of the TWW. Samples were withdrawn at three-day intervals, centrifuged at 3500 rpm for 10 mins to pellet the cyanobacterial cells and the absorbance of the supernatant was measured at the pre-determined λ max . The percentage of TWW decolorization was calculated using the following equation (Javed et al., 2022). where, Abs (I) and Abs (F) are initial and final absorbance values of the sample, respectively. Analysis of pollutant removal efficiency Ammoniacal nitrogen (NH₄⁺) was analyzed by salicylate method (Method 10023) using HACH powder pillows. Nitrate (NO₃⁻) was measured by cadmium reduction method (Method 8039) using HACH powder pillows. COD was determined by reactor digestion method (Method 8000) using HACH COD digestion vials. The above procedures followed the stipulated protocols in standard methods for wastewater analysis (APHA, 2012). Samples were analyzed in a DR900 colorimeter (HACH, CO, USA) and removal efficiencies were calculated according to the following equation. The phosphate (PO 4 3- ) removal efficiency was determined by the ascorbic acid method with potassium hydrogen phosphate (dibasic) as the standard (APHA, 2012). where, Conc (I) and Conc (F) are initial and final concentration values (mg/L) of the sample, respectively. Phytotoxicity assay Phytotoxicity assay was performed to evaluate the toxicity of untreated and treated TWW. The test was carried out using green gram ( Vigna radiata) at laboratory conditions. The undamaged seeds of almost identical size were surface-sterilized using 2% sodium hypochlorite, washed thoroughly and soaked overnight in sterile distilled water. Four seeds were placed in sterilized petri plates layered with two Whatman qualitative (Grade 01) filter papers. Two milliliters of TWW treated with NE and SS were watered into each plate daily up to 5 days. Positive control was run by watering the seeds with distilled water while negative control was run by watering with untreated TWW. Each treatment was triplicated. Germination (%) was calculated according to the following equation while the growth of seedlings was observed by measuring the lengths of plumule/shoot, and radicle/root compared to the negative control using the ImageJ (Image processing and analysis in Java) (version 1.53t) software. Harvesting of TWW-grown cyanobacterial biomass for lipid analysis Fresh biomass of NE and SS grown in 100% TWW was harvested after 28 days of initial culturing by the filtration method. The harvested biomass was transferred in to aluminum trays and oven-dried at 55°C until they were fully dried. The dried biomass was made into a fine powder, measured and stored in the freezer (-4˚C) for lipid analysis. Lipid extraction and quantification Lipid extraction was done in a Soxhlet apparatus (Biobase®, China) using hexane as the extraction solvent. Two grams of dried TWW-grown biomass of NE and SS was taken and wrapped in a Whatman No. 1 qualitative filter paper. It was refluxed with 50 ml of hexane for six hours with a water condenser. The hexane layer containing the extracted lipids was collected and vacuum-dried in a rotary evaporator (Heidolph™, Germany) at 37 ˚C to evaporate the solvent. The dried lipid was weighed and the total lipid content was calculated gravimetrically on w/w % of dry biomass by taking the difference in the pre- and final weights of the vial. For each treatment, three replicates were maintained. Fatty acid profile analysis Fatty acid methyl esters (FAMEs)/ biodiesel were prepared from extracted lipids following an acid-base transesterification reaction as follows; 200 µl of lipids were reacted with 2 ml of methanol containing 2% (w/v) NaOH for 15 min at 65 ºC in a water bath. Then, 2 ml of methanol containing 5% (v/v) H 2 SO 4 was added to the screw-capped glass tube and heated for another 15 min at 65 ºC. Esterified fatty acids were extracted into 1 ml of hexane by centrifugation (1500 rpm for 10 mins) (Javed et al., 2022). FAMEs were analyzed using a gas chromatograph (Agilent 7890B, CA, USA) equipped with a flame ionization detector (FID) in a capillary column (100 m×0.25 mm×0.2 μm) (Agilent J & W CP-Sil 88 for FAME, CA, USA). Ultra-high purity nitrogen gas (99.9995%) was used as the carrier gas. Initial column temperature was set at 100 °C, which was progressively raised to 180 °C at 8 °C/min, and then to 230 °C at 1 °C/min. The injector was retained at 260 °C with an injection volume of 1 μL in a splitless mode. The peaks were identified by comparing the retention times with those of a mixture of external standard methyl esters (Supelco® 37 FAME Mix, Sigma Aldrich, St. Louis, MO, USA). The results were expressed as the relative percentage of total peak area of FAMEs considered. Statistical Analysis Data were presented as mean ± standard deviation. The statistical analysis of the data was performed using analysis of variance (ANOVA) and when significant effects were detected, the groups were compared using a post-hoc Tukey’s HSD test. The level of significance used for all statistical tests is 5% (p < 0.05). The statistical program used was Minitab version 19.2. Results & Discussion Characterization of textile wastewater The pH, electric conductivity (EC), salinity and total dissolved solids (TDS) were recorded as 9.93, 704 mS, 350 mg/L and 470 mg/L, respectively. The initial characteristics of the TWW revealed a high COD value of 3046 mg/L, along with higher concentrations of phosphates (168 mg/L), ammoniacal nitrogen (70 mg/L), and nitrates (200 mg/L) (Table 1). These findings highlighted the necessity for adequate treatment of TWW before discharge into the environment. Cyanobacterial growth in textile wastewater The growth patterns of NE and SS in TWW were assessed by measuring optical density (OD) at 680 nm (Fig. 1), a standard wavelength for monitoring cyanobacterial growth and chlorophyll content. Both strains exhibited a gradual increase in OD values over the cultivation period, indicating successful adaptation and proliferation in the wastewater environment. Among the two, NE showed the most promising growth, achieving a growth rate of 90.82% ± 0.09, consistently displaying a higher OD increase compared to SS, which recorded a growth rate of 83.57% ± 0.06. The observed growth trends highlighted the adaptive capabilities of cyanobacteria in TWW, demonstrating their ability to utilize TWW as a nutrient source while tolerating its toxic components. Cyanobacteria are known for their resilience to environmental stressors and efficient uptake of inorganic nutrients (e.g., ammoniacal nitrogen, nitrates and phosphates) from industrial effluents, promoting biomass accumulation (Cai et al., 2013). The higher growth rate of NE (a heterocystous filamentous cyanobacterium) may be attributed to its nitrogen-fixing ability, allowing it to thrive even in nitrogen-limited conditions commonly found in TWW. Conversely, SS is a non-heterocystous cyanobacterium that relies on available nitrogen sources for growth, which may explain its relatively lower but still significant growth rate. Inorganic phosphates presented in the TWW also could have contributed to the notable algal growth, playing a key role in their metabolism (Mouga et al., 2024). These findings suggest that TWW could serve as a low-cost, sustainable cultivation medium for cyanobacterial biomass production, reducing the need for synthetic growth media while simultaneously supporting bioremediation applications. Further optimization of cultivation parameters, such as light intensity, aeration, and supplementation with additional nutrients, could enhance the growth efficiency of these strains in industrial-scale applications. Decolorization of textile wastewater Decolorization of TWW by NE and SS were evaluated by measuring OD at 775 nm. Significant color removal of TWW was observed from NE and SS with 79.03% ± 0.09 and 82.67% ± 0.09, respectively (Fig. 2). These results indicated that both strains possess strong biosorption and biodegradation capabilities, contributing to dye removal from TWW. The observed decolorization can be attributed to several mechanisms, including biosorption (cell surface adsorption), bioaccumulation (active transport of dye molecules in to the cell), extracellular polymeric substance (EPS) secretion, and enzymatic degradation (biodegradation). The negatively charged cell walls of cyanobacteria facilitate electrostatic interactions with dye molecules, allowing passive dye adsorption, while intracellular metabolic processes and oxidative enzymes (e.g., azoreductases, laccases, peroxidases) further contribute to biodegradation (Dudeja et al., 2025; Mishra & Maiti, 2019; Sudarshan et al., 2023). Chemical Oxygen Demand (COD) removal efficiency The NE and SS significantly reduced the initial COD value of 3042 mg/L to 107 mg/L and 210 mg/L, achieving COD removal efficiencies of 96.49% and 93.11%, respectively. This directly implied their ability to successfully consume organic matter present in TWW suggesting their capability to bioremediate TWW with high COD levels. A high COD level indicates a complex mixture of organic pollutants, which may require extended treatment time to remediate (Nguyen et al., 2022). Since COD is an indirect measure of organic compound concentration, assessing its reduction is crucial before TWW is discharged into the environment. According to Selvaraj & Arivazhagan, (2024), there is a strong correlation between high biomass productivity and COD removal efficiency since cyanobacteria utilize organic carbon as an energy source and a nutrient substrate for their growth. This ultimately improves the wastewater quality into permissible levels and it was evident in this study. The effective COD utilization by NE and SS can be attributed to factors such as species-specific metabolic activity, growth conditions, and nutrient availability in TWW. Additionally, oxygen generation by these photosynthetic prokaryotes enhances the biological degradation of organic matter, further contributing to COD reduction in TWW (Selvaraj & Arivazhagan, 2024). In a similar study, Javed et al., (2022) observed a COD removal of 59% by Chlorella vulgaris on unsterilized, 100% TWW, which was lower than the COD removal efficiencies observed in this study using Nostoc sp. and Spirulina sp. Brar et al., (2019) observed a 50% reduction of COD by Anaebaena ambigua in sterilized, 75% TWW. Raza et al., (2024) observed a COD reduction of 91.5% by a consortium of Chlorella vulgaris and Staphylococcus sp. Nutrient removal efficiency There was a significant reduction in the concentration of ammoniacal nitrogen, nitrate and phosphate present in the TWW after the phycoremediation experiment. NE achieved a 76.28% ammoniacal nitrogen removal, while SS exhibited a higher removal of 96.86%. The nitrate reduction was recorded as 69% and 73% with NE and SS, respectively. Meanwhile, the phosphate removal was detected as 81.14% with NE and 33.33% with SS. Overall, SS demonstrated a superior nitrogen removal efficiency, whereas NE was more efficient in phosphate removal. All these findings highlighted the potential of selected cyanobacterial species for nutrient removal from TWW. These reductions in nutrient levels corresponded with an increase in algal biomass, demonstrating a direct relationship between nutrient uptake and growth. However, some studies indicated that phosphorus uptake by cyanobacteria may not always be efficient, depending on species and environmental conditions (Tsolcha et al., 2017), which is also evident by SS in this study. Phosphorus (P) and Nitrogen (N) are key pollutants present in wastewater, posing significant threats to aquatic ecosystems, leading to eutrophication (Preisner et al., 2021). Cyanobacteria play a crucial role in nutrient removal from wastewater through N assimilation and phosphorylation. These two processes facilitate the uptake of N and P, which are vital for algal metabolism, growth, and biomass production. They assimilate N by converting inorganic N sources: nitrate (NO₃⁻), nitrite (NO₂⁻), and ammonium (NH₄⁺) into organic forms required for the synthesis of peptides, proteins, enzymes, chlorophylls, and genetic materials (RNA and DNA) via enzymatic reduction (Inabe et al., 2021; Raza et al., 2024). Among the different N sources, ammonium (NH₄⁺) is preferred by cyanobacteria due to its lower energy requirement for assimilation. Wastewater rich in ammonium, therefore, serves as a promising medium for cyanobacteria cultivation (Nguyen et al., 2022; Znad et al., 2018). Phosphorus is another essential macronutrient required for the production of lipids, proteins, nucleic acids, and ATP (adenosine triphosphate). Phosphate assimilation by cyanobacteria through phosphorylation is the primary mechanism for phosphate removal in wastewater (Znad et al., 2018). Inorganic phosphorus exists primarily in the forms of H₂PO₄⁻ (phosphoric acid) and HPO₄²⁻ (hydrogen phosphate). These forms are absorbed by cyanobacteria through phosphorylation, where phosphate is incorporated into intracellular organic compounds via specialized phosphate transporters in the plasma membrane (Burford et al., 2023; Markou et al., 2014) Various environmental factors affect the nutrient uptake rates in different cyanobacterial species, including initial nutrient concentration, light intensity, airflow rate, extracellular pH, temperature, and inoculum size (Tsolcha et al., 2017). Due to their diverse physiological and morphological traits, different species of cyanobacteria demonstrate varied effectiveness in removing N and P from wastewater effluents. Hence, the selection of an appropriate algal strain is crucial for optimizing nutrient removal efficiency. Based on a study by Brar et al., (2019), a nitrate removal of 52.95% and phosphate removal of 63.05% were achieved on sterilized, 75% TWW by Anaebaena ambigua. Raza et al., (2024) observed increased removal efficiencies by a Chlorella vulgaris-Staphylococcus consortium achieving NO 3 - (58.57%), and PO 4 3− (89.19%), compared to algae only treatment. Table 1. Physicochemical characteristics of textile wastewater before and after treatment with Nostoc ellipsosporum and Spirulina subsalsa Initial assessment of TWW TWW treated with Nostoc ellipsosporum TWW treated with Spirulina subsalsa Characteristic of TWW Unit Before treatment After treatment Removal efficiency (%) After treatment Removal efficiency (%) pH 9.93 8.54 8.62 Electric conductivity (EC) μS 704 906 1677 Salinity mg/L 350 457 839 Total dissolved solids (TDS) mg/L 470 375 20.2 390 17.02 Chemical oxygen demand (COD) mg/L 3046 107 96.49 210 93.11 Total phosphate (PO 4 3- ) mg/L 168 31.68 81.14 112 33.33 Ammoniacal nitrogen (NH 4 + ) mg/L 70 16.6 76.28 2.2 96.86 Nitrate (NO 3 - ) mg/L 200 62 69 146 73 Effect of cyanobacteria-treated textile wastewater on seed germination and seedling growth The effects of tap water (positive control), untreated TWW (negative control) and treated TWW by NE (NE-TWW) and SS (SS-TWW) on seed germination and seedling growth are depicted in Table 2. Seeds in the positive control, NE-TWW and SS-TWW treatments exhibited a 100% germination rate, whereas the negative control had a significantly lower germination rate of 25%. The initial physicochemical analysis revealed that TWW was toxic in nature due to the excess pollutant concentrations as shown in the Table 1. It was further confirmed by having an inhibitory effect on seed germination and seedling growth. After phycoremediation, toxicity of TWW was significantly reduced and showed improved seed germination indicating their pollutant removal capability. A seed was deemed to have successfully germinated if its root system were visible and measurable, with a minimum length of 1 mm. Seeds that did not germinate were assigned a root length of zero (Rekik et al., 2017). In this study, shoot length was highest in the positive control (8.12 cm), followed by NE-TWW (6.88 cm) and SS-TWW (6.54 cm), while the negative control exhibited the shortest shoot length (1.99 cm). Similarly, root length followed the same trend, with the positive control (6.44 cm) having the longest roots, followed by NE-TWW (4.97 cm) and SS-TWW (4.24 cm), whereas the negative control had the shortest root length (1.02 cm). These findings are in accordance with a similar study conducted by Selvaraj & Arivazhagan, (2024), which had maximum shoot and root lengths of approximately 8.5 cm and 5 cm, after 05 days irrigated with TWW treated with Spirulina platensis . Elakbawy et al., (2022)reported a shoot length of 5.69 cm on tomato seedlings when testing the effect of Nostoc muscorum crude extracts on treated sewage wastewater. Cyanobacteria release secondary metabolites to the medium during their growth, including plant growth hormones, micronutrients, macronutrients, and vitamins (Elakbawy et al., 2022). The release of these beneficial compounds by cyanobacteria also could be one of the reasons for the enhanced shoot and root development observed in the present study. These results suggest that the phycoremediated TWW enhances the germination process by providing additional nutrients to the medium, highlighting the potential use of phycoremediated TWW for irrigation. The reason for reduced seed growth in untreated TWW could be due to the excess nutrient concentrations, high levels of COD, and heavy metals which retard seedling growth by affecting water absorption and other metabolic activities (Dhaouefi et al., 2019). Different crop plant species also differ widely in response to different concentrations of wastewater with respect to seed germination, seedling growth and productivity (Fendri et al., 2013). Table 2. Phytotoxicity results of treatments showing germination (%), shoot length and root length Treatment Tap water TWW NE-TWW SS-TWW Germination (%) 100 a 25 b 100 a 100 a Shoot length (cm) 8.12 a 1.99 c 6.88 b 6.54 b Root length (cm) 6.44 a 1.02 c 4.97 b 4.24 b TWW: Textile wastewater, NE-TWW: Nostoc -treated TWW, SS-TWW: Spirulina -treated TWW Means followed by different superscript letters within a row are significantly different at p < 0.05. Total lipid content of textile wastewater-grown cyanobacterial biomass High lipid content in cyanobacterial cells is a key factor determining their potential for biodiesel production (Hawrot-Paw et al., 2021; Nagappan et al., 2020). The total lipid contents of the TWW-grown cyanobacterial strains are shown in Fig. 3. It is worth mentioning that, NE and SS demonstrated significant lipid yields of 21.5% and 25%, respectively, compared to several cyanobacterial strains used in TWW bioremediation (Mathimani et al., 2024). To the best of our knowledge, this is the first report that the lipid content and lipid profile of NE and SS grown in unsterilized and undiluted TWW have been analysed. These results suggest that both strains can thrive in unsterilized-raw TWW, utilizing it as a favorable growth medium while achieving enhanced lipid production. The variation in lipid content among two species could be a result from differences in their morphology, biochemical composition, and native environmental adaptations. Moreover, lipid profile and productivity are also influenced by multiple factors, including cultivation conditions, biomass harvesting, cell disruption methods, strain selection, solvent polarity, and extraction techniques (Fazal et al., 2018; Javed et al., 2022). Higher lipid accumulation in algae under wastewater conditions is attributed to nutrient limitation, which enhance lipid synthesis (Feng et al., 2011; Mahmood et al., 2023). Specifically, the nitrogen (N) and phosphorus (P) levels in wastewater may be lower than those in standard growth media (i.e., BG-11, Zarrouk), inducing nutrient starvation that triggers lipid accumulation (Purba et al., 2022). Mostafa et al., (2012) reported lipid contents of 15.5% and 7.3% for Nostoc humifusum and Spirulina platensis , respectively, when cultivated in domestic wastewater for biodiesel production. Hossain et al., (2018) reported a lipid content of 15.43% in Nostoc sp. cultured in half-strength BG-11 medium, while Nagappan et al., (2020) observed a lipid content of 15.7% in Nostoc sp. MCC41 when grown in BG-11 medium. Ambrozova et al., (2014) reported a lipid content of 13.41% for Spirulina platensis cultivated in BG-11, whereas Haberle et al., (2020) obtained a lipid content of 15.3% for Spirulina subsalsa grown in oil refinery wastewater. These findings highlighted the notable lipid yields achieved in TWW-grown NS and SS in this study, reinforcing their potential as viable feedstocks for biodiesel production. Fatty acid methyl ester (FAME) analysis for biodiesel production Characterization of FAME profile is crucial to determine the biodiesel quality of cyanobacteria (Mathimani et al., 2024). Therefore, the fatty acid composition of TWW-grown NE and SS was analyzed using GC-FID. In algal FAME profiles, fatty acids with carbon chain lengths of C16–C18 are the most commonly identified, as also confirmed by the results of the present study. The key FAMEs that determine biodiesel quality include palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and low concentrations of linoleic acid (C18:2), and linolenic acid (C18:3) (Hawrot-Paw et al., 2021). According to Knothe, (2005), two critical factors influencing biodiesel quality are oxidative stability and low-temperature performance. In the present study, both cyanobacterial strains exhibited a similar fatty acid composition, though slight variations were observed in the percentages of each fatty acid. The key FAMEs identified in both cyanobacterial strains are, palmitic acid (C16:0), oleic acid (C18:1), stearic acid (C18:0), lauric acid (C12:0), and myristic acid (C14:0) (Table 3). Notably, palmitic acid and oleic acid together accounted for more than 70% of the dominant FAMEs recorded in both species. Similarly, Phormidium lucidum grown in oil refinery wastewater was investigated for biodiesel production and was found to have palmitic and oleic acids as the predominant fatty acids, comprising 64% of the total FAMEs (Haberle et al., 2020), which is consistent with our findings. Cyanobacterial lipids are rich in both saturated and unsaturated fatty acids. Unsaturated fatty acids (UFAs) enhance biodiesel’s cold flow and cloud point, preventing fuel solidification in cold weather. However, UFAs are more prone to oxidation and rancidification unlike saturated fatty acids (SFAs). In contrast, SFAs contribute to crystal formation at low temperatures, leading to poor cold flow properties (Sadvakasova et al., 2021; Sinha et al., 2016). Since UFAs improve low-temperature performance, while SFAs enhance oxidative stability, a well-balanced FAME composition is essential for optimal biodiesel quality, specifically with low levels of UFAs. Among UFAs, monounsaturated fatty acids (MUFAs) are ideal for biodiesel production, whereas polyunsaturated fatty acids (PUFAs) are less favorable. Oleic acid (C18:1), a MUFA, is considered optimal due to its balanced oxidative stability and low-temperature performance (Feng et al., 2011; Knothe, 2005). High PUFA content can increase nitrogen oxide emissions, but a low PUFA concentration positively affects biodiesel’s flow properties, particularly at low temperatures (Hawrot-Paw et al., 2021). In this study, palmitic acid (C16:0) was the most abundant fatty acid, followed by oleic acid (C18:1) in both strains. NE contained 45.92% (C16:0) and 24.77% (C18:1), while SS had 45.88% (C16:0) and 28.13% (C18:1). As shown in Fig. 3, the SFA and MUFA percentages in both strains was optimal (Fig. 3), suggesting their suitability for third-generation biodiesel production. Table 3. Most dominant fatty acid methyl esters present in the textile wastewater-grown cyanobacterial biomass Name of fatty acid methyl ester Carbon number Fatty acid percentage (%) in NE Fatty acid percentage (%) in SS Palmitic acid C16:0 45.92 45.88 Oleic acid C18:1 (cis (n9) 24.77 28.13 Stearic acid C18:0 8.82 5.63 Cis-10-pentadecanoic acid C15:1 8.65 - Cis-10-heptadecanoic acid C17:1 - 8.58 Lauric acid C12:0 6.66 6.64 Myristic acid C14:0 5.19 5.14 Conclusion This study demonstrated the dual potential of NE and SS in TWW bioremediation and biodiesel production. Both cyanobacterial species exhibited significant growth in undiluted and unsterilized TWW, effectively removing pollutants while achieving high decolorization efficiencies. NE and SS exhibited notable COD removal efficiencies of 96.48% and 93.11%, respectively. SS showed superior ammoniacal nitrogen removal (96.86%), whereas NE exhibited better phosphate removal (81.14%) and nitrate removal (73%). The treated wastewater significantly improved seed germination and seedling growth, highlighting its potential application for irrigation. Furthermore, the TWW-grown cyanobacterial biomass demonstrated high lipid accumulation, with SS yielding 25% total lipids and NE yielding 21.5%. Fatty acid profiling confirmed the presence of key biodiesel precursors, such as palmitic acid (C16:0) and oleic acid (C18:1), indicating their suitability for sustainable biofuel production. This integrated approach presents a cost-effective and environmentally friendly alternative for TWW treatment, while simultaneously producing biodiesel from wastewater-grown biomass. Declarations Funding The authors declare that no funding was received for this study. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by S.M.D.C. Bandara. The first draft of the manuscript was written by S.M.D.C. Bandara and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Ambrozova, J. V., Misurcova, L., Vicha, R., Machu, L., Samek, D., Baron, M., Mlcek, J., Sochor, J., & Jurikova, T. (2014). Influence of extractive solvents on lipid and fatty acids content of edible freshwater algal and seaweed products, the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. Molecules , 19 (2), 2344–2360. https://doi.org/10.3390/molecules19022344 APHA, 2012. Standard Methods for the Examination of Water and Wastewater, twenty- second ed. American Public Health Association, Washington, DC. Bharagava, R. N., & Bhimrao, B. (2018). Textile Industry Wastewater: Environmental and Health Hazards and Treatment Approaches . https://www.researchgate.net/publication/328701616 Brar, A., Kumar, M., Vivekanand, V., & Pareek, N. (2019). Phycoremediation of textile effluent-contaminated water bodies employing microalgae: nutrient sequestration and biomass production studies. International Journal of Environmental Science and Technology , 16 (12), 7757–7768. https://doi.org/10.1007/s13762-018-2133-9 Burford, M. A., Willis, A., Xiao, M., Prentice, M. J., & Hamilton, D. P. (2023). Understanding the relationship between nutrient availability and freshwater cyanobacterial growth and abundance. Inland Waters , 13 (2), 143–152. https://doi.org/10.1080/20442041.2023.2204050 Cai, T., Park, S. Y., & Li, Y. (2013). Nutrient recovery from wastewater streams by microalgae: Status and prospects. In Renewable and Sustainable Energy Reviews (Vol. 19, pp. 360–369). https://doi.org/10.1016/j.rser.2012.11.030 Cuellar-Bermudez, S. P., Aleman-Nava, G. S., Chandra, R., Garcia-Perez, J. S., Contreras-Angulo, J. R., Markou, G., Muylaert, K., Rittmann, B. E., & Parra-Saldivar, R. (2017). Nutrients utilization and contaminants removal. A review of two approaches of algae and cyanobacteria in wastewater. Algal Research , 24 , 438–449. https://doi.org/10.1016/j.algal.2016.08.018 Dhaouefi, Z., Toledo-Cervantes, A., Ghedira, K., Chekir-Ghedira, L., & Muñoz, R. (2019). Decolorization and phytotoxicity reduction in an innovative anaerobic/aerobic photobioreactor treating textile wastewater. Chemosphere , 234 , 356–364. https://doi.org/10.1016/j.chemosphere.2019.06.106 Dudeja, C., Masroor, S., Mishra, V., Kumar, K., Sansar, S., Yadav, P., Chaturvedi, N., Singh, R., & Kumar, A. (2025). Cyanobacteria-based bioremediation of environmental contaminants: advances and computational insights. Discover Agriculture , 3 (1), 42. https://doi.org/10.1007/s44279-025-00193-9 Elakbawy, W. M., Shanab, S. M. M., & Shalaby, E. A. (2022). Enhancement of plant growth regulators production from microalgae cultivated in treated sewage wastewater (TSW). BMC Plant Biology , 22 (1). https://doi.org/10.1186/s12870-022-03764-w El-Sheekh, M., El-Dalatony, M. M., Thakur, N., Zheng, Y., & Salama, E. S. (2022). Role of microalgae and cyanobacteria in wastewater treatment: genetic engineering and omics approaches. In International Journal of Environmental Science and Technology (Vol. 19, Issue 3, pp. 2173–2194). Springer Science and Business Media Deutschland GmbH. https://doi.org/10.1007/s13762-021-03270-w El-Sheekh, M. M., Galal, H. R., Mousa, A. S. H., & Farghl, A. A. M. (2023). Coupling wastewater treatment, biomass, lipids, and biodiesel production of some green microalgae. Environmental Science and Pollution Research , 30 (12), 35492–35504. https://doi.org/10.1007/s11356-023-25628-y Fazal, T., Mushtaq, A., Rehman, F., Ullah Khan, A., Rashid, N., Farooq, W., Rehman, M. S. U., & Xu, J. (2018). Bioremediation of textile wastewater and successive biodiesel production using microalgae. In Renewable and Sustainable Energy Reviews (Vol. 82, pp. 3107–3126). Elsevier Ltd. https://doi.org/10.1016/j.rser.2017.10.029 Fendri, I., Ben Saad, R., Khemakhem, B., Ben Halima, N., Gdoura, R., & Abdelkafi, S. (2013). Effect of treated and untreated domestic wastewater on seed germination, seedling growth and amylase and lipase activities in Avena sativa L. Journal of the Science of Food and Agriculture , 93 (7), 1568–1574. https://doi.org/10.1002/jsfa.5923 Feng, D., Chen, Z., Xue, S., & Zhang, W. (2011). Increased lipid production of the marine oleaginous microalgae Isochrysis zhangjiangensis (Chrysophyta) by nitrogen supplement. Bioresource Technology , 102 (12), 6710–6716. https://doi.org/10.1016/j.biortech.2011.04.006 Haberle, I., Hrustić, E., Petrić, I., Pritišanac, E., Šilović, T., Magić, L., Geček, S., Budiša, A., & Blažina, M. (2020). Adriatic cyanobacteria potential for cogeneration biofuel production with oil refinery wastewater remediation. Algal Research , 50 . https://doi.org/10.1016/j.algal.2020.101978 Hasan, M. N., Altaf, M. M., Khan, N. A., Khan, A. H., Khan, A. A., Ahmed, S., Kumar, P. S., Naushad, M., Rajapaksha, A. U., Iqbal, J., Tirth, V., & Islam, S. (2021). Recent technologies for nutrient removal and recovery from wastewaters: A review. Chemosphere , 277 . https://doi.org/10.1016/j.chemosphere.2021.130328 Hawrot-Paw, M., Ratomski, P., Koniuszy, A., Golimowski, W., Teleszko, M., & Grygier, A. (2021). Fatty acid profile of microalgal oils as a criterion for selection of the best feedstock for biodiesel production. Energies , 14 (21). https://doi.org/10.3390/en14217334 Hossain, T., Miah, A. B., Mahmud, S. A., & Mahin, A. Al. (2018). Enhanced Bioethanol Production from Potato Peel Waste Via Consolidated Bioprocessing with Statistically Optimized Medium. Applied Biochemistry and Biotechnology , 186 (2), 425–442. https://doi.org/10.1007/s12010-018-2747-x Inabe, K., Miichi, A., Matsuda, M., Yoshida, T., Kato, Y., Hidese, R., Kondo, A., & Hasunuma, T. (2021). Nitrogen availability affects the metabolic profile in cyanobacteria. Metabolites , 11 (12). https://doi.org/10.3390/metabo11120867 Javed, F., Rehman, F., Khan, A. U., Fazal, T., Hafeez, A., & Rashid, N. (2022). Real textile industrial wastewater treatment and biodiesel production using microalgae. Biomass and Bioenergy , 165 . https://doi.org/10.1016/j.biombioe.2022.106559 Khan, S., & Malik, A. (2014). Environmental and health effects of textile industry wastewater. In Environmental Deterioration and Human Health: Natural and Anthropogenic Determinants (Vol. 9789400778900, pp. 55–71). Springer Netherlands. https://doi.org/10.1007/978-94-007-7890-0_4 Khatri, J., Nidheesh, P. V., Anantha Singh, T. S., & Suresh Kumar, M. (2018). Advanced oxidation processes based on zero-valent aluminium for treating textile wastewater. Chemical Engineering Journal , 348 , 67–73. https://doi.org/10.1016/j.cej.2018.04.074 Kishor, R., Purchase, D., Ferreira, L. F. R., Mulla, S. I., Bilal, M., & Bharagava, R. N. (2020). Environmental and Health Hazards of Textile Industry Wastewater Pollutants and Its Treatment Approaches. In Handbook of Environmental Materials Management (pp. 1–24). Springer International Publishing. https://doi.org/10.1007/978-3-319-58538-3_230-1 Knothe, G. (2005). Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Processing Technology , 86 (10), 1059–1070. https://doi.org/10.1016/j.fuproc.2004.11.002 Mahmood, T., Hussain, N., Shahbaz, A., Mulla, S. I., Iqbal, H. M. N., & Bilal, M. (2023). Sustainable production of biofuels from the algae-derived biomass. Bioprocess and Biosystems Engineering , 46 (8), 1077–1097. https://doi.org/10.1007/s00449-022-02796-8 Markou, G., Vandamme, D., & Muylaert, K. (2014). Microalgal and cyanobacterial cultivation: The supply of nutrients. In Water Research (Vol. 65, pp. 186–202). Elsevier Ltd. https://doi.org/10.1016/j.watres.2014.07.025 Mathimani, T., Alshiekheid, M. A., Sabour, A., Le, T. H. T., & Xia, C. (2024). Appraising the phycoremediation potential of cyanobacterial strains Phormidium and Oscillatoria for nutrient removal from textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass. Environmental Research , 241 . https://doi.org/10.1016/j.envres.2023.117628 Mishra, S., & Maiti, A. (2019). Applicability of enzymes produced from different biotic species for biodegradation of textile dyes. In Clean Technologies and Environmental Policy (Vol. 21, Issue 4, pp. 763–781). Springer Verlag. https://doi.org/10.1007/s10098-019-01681-5 Mona, S., Kumar, V., Deepak, B., & Kaushik, A. (2020). Cyanobacteria: The Eco-Friendly Tool for the Treatment of Industrial Wastewaters. In Bioremediation of Industrial Waste for Environmental Safety (pp. 389–413). Springer Singapore. https://doi.org/10.1007/978-981-13-3426-9_16 Mostafa, S. S. M., Shalaby, E. A., & Mahmoud, G. I. (n.d.). Cultivating Microalgae in Domestic Wastewater for Biodiesel Production. Not Sci Biol , 2012 (1), 56–65. www.notulaebiologicae.ro Mouga, T., Pereira, J., Moreira, V., & Afonso, C. (2024). Unveiling the Cultivation of Nostoc sp. under Controlled Laboratory Conditions. Biology , 13 (5). https://doi.org/10.3390/biology13050306 Nagappan, S., Bhosale, R., Duc Nguyen, D., Pugazendhi, A., Tsai, P. C., Chang, S. W., Ponnusamy, V. K., & Kumar, G. (2020). Nitrogen-fixing cyanobacteria as a potential resource for efficient biodiesel production. Fuel , 279 . https://doi.org/10.1016/j.fuel.2020.118440 Nguyen, L. N., Aditya, L., Vu, H. P., Johir, A. H., Bennar, L., Ralph, P., Hoang, N. B., Zdarta, J., & Nghiem, L. D. (2022). Nutrient Removal by Algae-Based Wastewater Treatment. Current Pollution Reports , 8 (4), 369–383. https://doi.org/10.1007/s40726-022-00230-x Preisner, M., Neverova-Dziopak, E., & Kowalewski, Z. (2021). Mitigation of eutrophication caused by wastewater discharge: A simulation-based approach. Ambio , 50 (2), 413–424. https://doi.org/10.1007/s13280-020-01346-4 Purba, L. D. A., Othman, F. S., Yuzir, A., Mohamad, S. E., Iwamoto, K., Abdullah, N., Shimizu, K., & Hermana, J. (2022). Enhanced cultivation and lipid production of isolated microalgae strains using municipal wastewater. Environmental Technology and Innovation , 27 . https://doi.org/10.1016/j.eti.2022.102444 Raza, N., Rizwan, M., & Mujtaba, G. (2024). Bioremediation of real textile wastewater with a microalgal-bacterial consortium: an eco-friendly strategy. Biomass Conversion and Biorefinery , 14 (6), 7359–7371. https://doi.org/10.1007/s13399-022-03214-5 Rekik, I., Chaabane, Z., Missaoui, A., Bouket, A. C., Luptakova, L., Elleuch, A., & Belbahri, L. (2017). Effects of untreated and treated wastewater at the morphological, physiological and biochemical levels on seed germination and development of sorghum (Sorghum bicolor (L.) Moench), alfalfa (Medicago sativa L.) and fescue (Festuca arundinacea Schreb.). Journal of Hazardous Materials , 326 , 165–176. https://doi.org/10.1016/j.jhazmat.2016.12.033 Sadvakasova, A. K., Kossalbayev, B. D., Zayadan, B. K., Kirbayeva, D. K., Alwasel, S., & Allakhverdiev, S. I. (2021). Potential of cyanobacteria in the conversion of wastewater to biofuels. In World Journal of Microbiology and Biotechnology (Vol. 37, Issue 8). Springer Science and Business Media B.V. https://doi.org/10.1007/s11274-021-03107-1 Selvaraj, D., & Arivazhagan, M. (2024). Synergistic effects of Spirulina platensis cultivation in textile wastewater towards nutrient removal and seed germination study. Environmental Pollution , 357 . https://doi.org/10.1016/j.envpol.2024.124435 Sinha, S. K., Gupta, A., & Bharalee, R. (2016). Production of biodiesel from freshwater microalgae and evaluation of fuel properties based on fatty acid methyl ester profile. Biofuels , 7 (1), 105–121. https://doi.org/10.1080/17597269.2015.1118781 Stanier, R. Y., Kunisawa, R., Mandel, M., & Cohen-Bazire, G. (1971). Purification and Properties of Unicellular Blue-Green Algae (Order Chroococcales). In BACTEROLOGICAL REVIEWS . Sudarshan, S., Harikrishnan, S., RathiBhuvaneswari, G., Alamelu, V., Aanand, S., Rajasekar, A., & Govarthanan, M. (2023). Impact of textile dyes on human health and bioremediation of textile industry effluent using microorganisms: current status and future prospects. In Journal of Applied Microbiology (Vol. 134, Issue 2). Oxford University Press. https://doi.org/10.1093/jambio/lxac064 Touliabah, H. E. S., El-Sheekh, M. M., Ismail, M. M., & El-Kassas, H. (2022). A Review of Microalgae-and Cyanobacteria-Based Biodegradation of Organic Pollutants. In Molecules (Vol. 27, Issue 3). MDPI. https://doi.org/10.3390/molecules27031141 Tsolcha, O. N., Tekerlekopoulou, A. G., Akratos, C. S., Aggelis, G., Genitsaris, S., Moustaka-Gouni, M., & Vayenas, D. V. (2017). Biotreatment of raisin and winery wastewaters and simultaneous biodiesel production using a Leptolyngbya-based microbial consortium. Journal of Cleaner Production , 148 , 185–193. https://doi.org/10.1016/j.jclepro.2017.02.026 Znad, H., Al Ketife, A. M. D., Judd, S., AlMomani, F., & Vuthaluru, H. B. (2018). Bioremediation and nutrient removal from wastewater by Chlorella vulgaris. Ecological Engineering , 110 , 1–7. https://doi.org/10.1016/j.ecoleng.2017.10.008 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6947833","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485627902,"identity":"8d72adc5-a405-4377-926b-cc86e4c75fcd","order_by":0,"name":"Chathurika Bandara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDACCQYGZiDFA6TZGBgqGHjYG8DiFgZ4tDA2I7ScYeDhOQARJ6iFAayFsQ2omZAW/tnNzx8Xtt2TkWw/wPbg57w7Mjz8Bxg//GCQMMZpyZ1jhs0z24p5pHkS2A17tz3j4ZFIYJbsYZAww6XFQCLBsJm3LYFHjiGBTYJ322Eee6ATpYFm2eDWkv4RooX/AZvk3zmHeYAOY/6NX0sOxBZpiQQ2ad4GoBagdSBbcDpM4kZO4Wyecwk8kjMethvLHANqkUhss+wxwO19/hnpGz7zlCXYS5xPPvbwTc1hex7+w4dv/KiwMWzApQcBGBuQGLgjchSMglEwCkYBEQAAYoBItdXy+xcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2192-8209","institution":"National Institute of Fundamental Studies","correspondingAuthor":true,"prefix":"","firstName":"Chathurika","middleName":"","lastName":"Bandara","suffix":""},{"id":485627903,"identity":"24c66ffe-5e1f-45c4-9d62-d55d2030a873","order_by":1,"name":"Yuansong Wei","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuansong","middleName":"","lastName":"Wei","suffix":""},{"id":485627904,"identity":"322535d8-825b-4acf-ac7f-fa184cfb3f63","order_by":2,"name":"Charmalie Abayasekara","email":"","orcid":"","institution":"University of Peradeniya","correspondingAuthor":false,"prefix":"","firstName":"Charmalie","middleName":"","lastName":"Abayasekara","suffix":""},{"id":485627905,"identity":"49630445-65ca-4ce6-81be-8a18c68f4a53","order_by":3,"name":"Renuka Ratnayake","email":"","orcid":"","institution":"National Institute of Fundamental Studies","correspondingAuthor":false,"prefix":"","firstName":"Renuka","middleName":"","lastName":"Ratnayake","suffix":""}],"badges":[],"createdAt":"2025-06-22 05:41:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6947833/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6947833/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86859962,"identity":"fc3d5963-9d95-4302-96de-3cabfa05cc81","added_by":"auto","created_at":"2025-07-16 11:58:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23402,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth performance of \u003cem\u003eNostoc\u003c/em\u003e \u003cem\u003eellipsosporum \u003c/em\u003e(NE)\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Spirulina subsalsa \u003c/em\u003e(SS)\u003cem\u003e \u003c/em\u003ein textile wastewater\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6947833/v1/cc7b0957d721272d392f99ce.png"},{"id":86859963,"identity":"597466ca-bbd4-407f-a867-d7490c81f2df","added_by":"auto","created_at":"2025-07-16 11:58:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23006,"visible":true,"origin":"","legend":"\u003cp\u003eTextile wastewater decolorization by \u003cem\u003eNostoc\u003c/em\u003e \u003cem\u003eellipsosporum \u003c/em\u003e(NE)\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Spirulina subsalsa \u003c/em\u003e(SS)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6947833/v1/4a72adb0b15c2c8ff397ded4.png"},{"id":86860306,"identity":"21d7744a-aa12-4af9-aed5-d267630990ee","added_by":"auto","created_at":"2025-07-16 12:06:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15144,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of total lipid content, MUFA, and SFA present in textile wastewater-grown \u003cem\u003eNostoc\u003c/em\u003e \u003cem\u003eellipsosporum \u003c/em\u003e(NE)\u003cem\u003e \u003c/em\u003eand\u003cem\u003eSpirulina subsalsa \u003c/em\u003e(SS)\u003cem\u003e \u003c/em\u003e(MUFA: Monounsaturated fatty acids, SFA: Saturated fatty acids)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6947833/v1/7d4a09e45905423d9fd0df57.png"},{"id":86861538,"identity":"eb8ad6d4-999f-428a-b0da-5a08c84b13ee","added_by":"auto","created_at":"2025-07-16 12:22:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1111935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6947833/v1/5d63ff56-0a1f-49dc-b98a-8b28c0680019.pdf"}],"financialInterests":"","formattedTitle":"Phycoremediation potential of Nostoc ellipsosporum and Spirulina subsalsa for pollutant removal from real textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe textile industry is one of the most water-intensive industries, consuming large volumes of water for different manufacturing processes like sizing, de-sizing, scouring, bleaching, mercerizing, dyeing, and printing (Brar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Khan \u0026amp; Malik, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), in turn generating significant amounts of wastewater. These wastewaters are typically highly-colored due to the high load of toxic dyes, and contain heavy metals, recalcitrant organic compounds, and excessive amounts of inorganic ions. Textile wastewater (TWW) is toxic, carcinogenic and mutagenic due to the presence of synthetic dyes such as azo, anthraquinone, and phthalocyanine dyes (Khan \u0026amp; Malik, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Khatri et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). They reduce light penetration and photosynthetic activity in water bodies, consequently limiting oxygen supply to aquatic organisms and inhibiting their growth (Kishor et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The discharge of nutrient-rich (nitrogen and phosphorous) wastewater is also a major source of water pollution owing to eutrophication. Thus, nutrient removal has become crucial alongside organic pollutant removal (Hasan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConventional wastewater treatment methods, such as chemical coagulation, electro-flocculation, adsorption, advanced oxidation processes, and membrane technology, often present challenges related to high costs, secondary pollution, sludge management and inefficiency in removing complex contaminants (Bharagava \u0026amp; Bhimrao, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mona et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a sustainable and cost-effective alternative, bioremediation of TWW using cyanobacteria has gained significant attention due to their ability to metabolize pollutants while simultaneously consuming them to produce valuable biomass. This biomass could be employed in generation of biofuels, biopolymers and biofertilizers etc (Sadvakasova et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCyanobacteria, also known as blue-green algae are prokaryotic photosynthetic microorganisms known for their rapid growth and high tolerance to extreme environmental conditions. They have demonstrated remarkable potential in the bioremediation of textile effluents (Mathimani et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Selvaraj \u0026amp; Arivazhagan, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As photosynthetic organisms, cyanobacteria can release O\u003csub\u003e2\u003c/sub\u003e while assimilating CO\u003csub\u003e2,\u003c/sub\u003e unlike bacteria-based wastewater treatment where a significant amount of CO\u003csub\u003e2\u003c/sub\u003e is emitted. Cyanobacteria employ several mechanisms in TWW bioremediation (Tsolcha et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These mechanisms include dye decolorization, biosorption, biomineralization, biotransformation, bioaccumulation and enzymatic biodegradation, while sequestering heavy metals, and assimilating inorganic and organic compounds, leading to improved water quality (Mona et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Touliabah et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, cyanobacteria accumulate high lipid contents under nutrient-stress conditions, making them a promising feedstock for biodiesel production (M. M. El-Sheekh et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hence, integrating TWW bioremediation with successive biodiesel production not only addresses environmental concerns but also enhances the economic feasibility of the process. However, the complexity of wastewater and algal ecological characteristics induce techno-economic challenges for its industrial implementation (Brar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nguyen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study explored the dual role of two filamentous cyanobacterial species, namely \u003cem\u003eNostoc ellipsosporum\u003c/em\u003e (NE) and \u003cem\u003eSpirulina subsalsa\u003c/em\u003e (SS) in TWW remediation and their potential for biodiesel generation. Limited studies have been conducted to explore the potential of cyanobacteria in TWW bioremediation, compared to other wastewater types (Cuellar-Bermudez et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; M. El-Sheekh et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To the best of our knowledge, this could be the first report of utilizing above-mentioned cyanobacterial species to treat unsterilized and undiluted (100%) TWW under greenhouse conditions without addition of any extra nutrients, with a key focus of investigating the practicability of this strategy, saving energy and freshwater usage, in terms of sterilization and dilution, respectively. The study aimed to evaluate the growth performance of NE and SS in TWW, while focusing on their ability to decolorize TWW. The study assessed their effectiveness in removing COD and inorganic nutrients (ammoniacal nitrogen, nitrate, phosphate) from TWW. Moreover, this study investigated the impact of treated TWW on seed germination, particularly focusing on shoot and root development. Furthermore, the research objectives were to explore the total lipid content of TWW-grown biomass while assessing their fatty acid profile for biodiesel production. Thus, this research aimed to establish an eco-friendly and cost-effective approach to TWW treatment while employing TWW-grown biomass for biodiesel production. The findings of this study could pave the way for a circular bioeconomy, where wastewater treatment and energy generation are seamlessly integrated for environmental and industrial sustainability.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003e\u003cstrong\u003eCyanobacterial species and collection of textile wastewater\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cyanobacterial cultures (NE and\u003cem\u003e\u0026nbsp;\u003c/em\u003eSS) were obtained from the NIFS-Sri Lanka culture collection (NIFS-SLCC), registered in the World Data Centre for Microorganisms (WDCM) (Reg. No: 1245) and maintained in BG-11 (Stanier et al., 1971) medium in 250 ml conical flasks at room temperature under 2500 lux of white fluorescent light till exponentially growing phase is attained. TWW was collected from a leading textile company in the Biyagama Export Processing Zone (6.9775\u0026deg; N, 79.9800\u0026deg; E), Sri Lanka and their initial physicochemical parameters were assessed in accordance with the established guidelines provided by American Public Health Association, (APHA, 2012). The pH, electric conductivity (EC), salinity, total dissolved solids (TDS) were measured onsite using a portable multiparameter (Oyster\u003csup\u003eTM\u003c/sup\u003e, NH, USA) (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of cyanobacterial growth and TWW decolorization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 10% (v/v) homogenous algal suspension was inoculated into transparent glass tanks containing a total volume of 20 L of unsterilized and undiluted (100%) TWW. They were maintained under greenhouse conditions with a photoperiod of 12:12 h employing continuous air bubbling into the tanks using aerator pumps. The growth of NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eSS\u0026nbsp;was monitored during three-day intervals by measuring optical density at 680 nm using an UV\u0026ndash;visible spectrophotometer (Agilent Carry 60, CA, USA) over a period of 28 days to monitor their growth. To assess the decolorization of the TWW by NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eSS, an UV-spectral scan was done initially within the visible range (400 nm to 800 nm) to find the maximum absorbance wavelength (\u0026lambda;\u003csub\u003emax\u003c/sub\u003e) of the TWW. Samples were withdrawn at three-day intervals, centrifuged at 3500 rpm for 10 mins to pellet the cyanobacterial cells and the absorbance of the supernatant was measured at the pre-determined \u0026lambda;\u003csub\u003emax\u003c/sub\u003e. The percentage of TWW decolorization was calculated using the following equation (Javed et al., 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"374\" height=\"61\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere, Abs (I) and Abs (F) are initial and final absorbance values of the sample, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of pollutant removal efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmmoniacal nitrogen (NH₄⁺) was analyzed by salicylate method (Method 10023) using HACH powder pillows. Nitrate (NO₃⁻)\u0026nbsp;was measured by cadmium reduction method (Method 8039) using HACH powder pillows. COD was determined by reactor digestion method (Method 8000) using HACH COD digestion vials. The above procedures followed the stipulated protocols in standard methods for wastewater analysis (APHA, 2012). Samples were analyzed in a DR900 colorimeter (HACH, CO, USA) and removal efficiencies were calculated according to the following equation. The phosphate (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e) removal efficiency was determined by the ascorbic acid method with potassium hydrogen phosphate (dibasic) as the standard (APHA, 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"473\" height=\"67\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere, Conc (I) and Conc (F) are initial and final concentration values (mg/L) of the sample, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhytotoxicity assay was performed to evaluate the toxicity of untreated and treated TWW. The test was carried out using green gram (\u003cem\u003eVigna radiata)\u003c/em\u003e at laboratory conditions. The undamaged seeds of almost identical size were surface-sterilized using 2% sodium hypochlorite, washed thoroughly and soaked overnight in sterile distilled water. Four seeds were placed in sterilized petri plates layered with two Whatman qualitative (Grade 01) filter papers. Two milliliters of TWW treated with NE and SS were watered into each plate daily up to 5 days. Positive control was run by watering the seeds with distilled water while negative control was run by watering with untreated TWW. Each treatment was triplicated. Germination (%) was calculated according to the following equation while the growth of seedlings was observed by measuring the lengths of plumule/shoot, and radicle/root compared to the negative control using the ImageJ (Image processing and analysis in Java) (version 1.53t) software.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAfcAAAA4CAYAAADgvLHyAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAABWCSURBVHhe7Z3rq1XFG8e3v/dmWq8iRLIgqUjiZCEZGJRdJBKKU9aLIDBPRWBFpSES3TxRBCFZUtCL6CT2QoLTTSjQkuwCRoVBGRHSqy5Wf4C/9ZmzvtvnjLP2Xmvv7dlr7/18YM6sPWvW3NaceWbNPDMz70RGw3Ecx3GcoeF/ue04juM4zpDgwt1xHMdxhgwX7o7jOI4zZLhwdxzHcZwhw4W74ziO4wwZLtwdx3EcZ8hw4e44NeS+++5rzJs3L5jPPvssdx0Mnn/++cb555/fTL/TfwbpfXz77beNRYsWhbRef/31uatTFRfuzshB46GGjkbkzz//zO/M8M477zTv0yj2g1deeaUxMTGR/xocnnjiicauXbsahw4dahw+fDh3HXzoYFEf6LgMIj///HNjbGws/1VvLr300sZPP/2U/3I6xYW7M3LQeExPT4frv//+u3H//feHa3H77bc3tmzZEq4RUv1iyZIl+dVgQCfp2WefbWzYsKFx1llnhXL2PbLqA+9kUBiktNYVF+7OSHLGGWcEe82aNY3du3efMvS9YMGCYHsjU54jR47kV8PHVVddFToqjz76aO5y+mAo2oejnW5x4e6MNJOTk8G+++67Txmed6px7Nix/MrpFOrgl19+mf9ynM5x4e6MNAwdI+CPHj3aeOONN3LXRmPx4sX51UmklGS/quz8vZ2Pvfzyy4Ob5vStghzz0vD+++83w8QmrCKskhphp/wy+sCUguLh2vojXt3D72uvvdYMs9VcMumPleTisNFTII/w2GOPBT+tvj6Jn/sKLzYijhvbppU06B7h/fLLL7PKALp9F+RNCl42blue+LfpVFosyovSgyEtuAPxXHHFFWGq6KOPPmr6wQjC5Bmlh7BIe0xcF/DHvHsZ4jSSJ5WT6DQdcb0R5N3Gib8UxGHrDfG3qmcjDQfHOM6oceDAASaDw/Uff/xxYunSpeH34cOHg5u9L/CH25o1a3KXGaanp4N71knIXWYYGxsL4U5MTJzIOg/BbXx8PPjF7dVXXw1uxLlw4cLg10J4io/0AH7xh3+FCUqDwuQe8eOPdIstW7bMih+TSruF+AlHacBOpUFl1ios4BmlgbRhuMZtamoq9zUDecAonlR69V4oW5UVz+Amun0XRe/YlqfKhzwoPULhkg69D+VZcQvc4joGpJsweC4uN8UNeg/yByo3TCuUdqWJOEmzTU/ZdJStk6rn8mfDi+PFLRWvcypeKs5IogZQqCFSYxLfF9aPkN+44ccf7jRKwja8Fvm1qNGLBV7cAAPCiIbTIn/2+bghBZ6L0y4URnxf7jYfReUQozTgX9BQ42YFouLg3VjIayx88YebhIbep+j2XRTlTXmxZQwIMFtP9LzNi4RVHCZu9lkhQaY8ghV4IlU+kMpXjPKjTi6QZpueKuloVydTzwncU2Vo667qjXMqPizvOBk33nhjIxMsYTiU4epect555+VXJ6miCX/uuefmVzPo96effhpshjmzRrJx2223hd9C/vbv3x9sy0UXXZRfNRpfffVVoaLYd999F+yVK1cGW2jYNB567hQpLi5atCjYsHfv3mDzbiwME5PfOG7cFQ7PZO1buLZ0+y6KiN/RihUrQl0SUsizeVFaPvnkk2C3g6HrTNjNUvJUGB9//HGwGeKnbKjLnaD3vHr16jAUT90izR9++GFwhzLpKFsnf/zxx2CvXbs22K1YtmxZI+s0NTZu3BimBBiiJw2p9+z4nLvjNGEZF43H5s2bG//++2/uWl+OHz8e7P/++y/YmuuWWbVqVXDvRgB/8803+VXvkADZsWNHc76ZxhrWr18fbFD+bJ4wEpq///57sAcF8krHkTlizc1XITUXrzAQpKAVC1rtURU6IdlXe+O6664L/w/Lly8P6dV7gjLpKFsnv//++2Br9UorEOSsf8++8kMH46abbgrlmJrDd1y4O04Tvj4ef/zx0Hi9/PLLuWt9OfPMM/OrGSYnJ8NXTGzsV1dVUl+63YIAIa185Z199tmh0Wf0YHp6OtyLSeUJk/JbVxBAF1xwQbDZoAjlNvJQFb6Y43KQgfnz5we7G1AyRXgiqHlPCPI777wzvztDu3SIdnWyaicEAU/5IeSnpqYaf/31VxhlsJ0PZwYX7o5jYHh6bGxs1pBqTNyQzPUSMMV3yy23BBshx4hD2eHdKtDQw8GDB4MtpBl9zTXXBLsKfLVt3749NNBq7BHu8fD7unXrgo3G9aDz3HPPhU4jgkkdplYjKilhxVA7y+RaCTLeF3Vhz549uctJymjL85Wud0s6+X9AkNv/hzLpKFsnr7zyymB/8MEHwRapskGLX1r7CHmmhtQZH+Y9FjrFhbszkmjYPSU4XnrppfzqVGjYvv7662YDiP3mm2+G63/++SfYQo2fbQQVb5HfVKO2adOmpjvpZQibDsi1114b3IBGjgaYYV+FxVciDaDNo+ItO+1w6623hrgQxgqHtGzbti003vfcc09wA3U64rzFvPvuu6FBbgdxEwf519AreeOr0i6VSpVzTMpPlXdR5LeoPCVIFZZ0CVRvyM8LL7wQruN0q47hh3vK6wMPPBDKbevWrc20YSP0MIJha56X7ghhIBQZwtbvVvBubX1DkNs5/LLpKFMn6YxQv3bu3DmrflE2S5cuPSWtsT86MdSRQRrFmTOyXrPjjBRoAlP1MVnDMEszWEgjOAa/aABzD21gNHelxYthaRRkjWHTTZrLPEt8cpfWr+LC2PSgucw9DGHoPr+tprLAv/xhSANpE8Rn44k1vIsgLuJU2lNpkBa0TKuwjx49Oiud1sThUhZxWZJP60fpwvBuYrp9Fxi5YZS3uDz13mzeFB/pzb6Am36VT/njtyAchcEzChfQXFf9w3CdKmvqofJH/gnDxm/DtJAnG75Nq6VsOtrVSSBsvaNU2agMeU55kFHenFOZx5+skJwRRj1l5q/4KuoF9NbR5qaX771qx8LIA8PwzLtajWu+alGSYp52LrZ5dZxhxoflRxyEOZqxUqLpFffee28Q7AypShPacYChVZZIWcEO7M7mOE5v6Eq4M39Cw818kpY6oJDB/E67eZ1+wXwN6bRzQ/1AZdVPKIs77rgjzBkjjC28Q71X3nHR+2TLyCLhzRc7X2doRUsRxnEW5gpfmkcHRo90Oh8KXI5TV6irtHnt2m9GL7Xkkba0lVys4rc0YXC+AzTHxpwHc2iArTmReF6lLmh+lLmgfkIaKKt+wZwWc1l2rk/wbrnH+2zlj/k55sji+bgYlXld64QztzBHSrtB3aFeYLj2+VOnztAe0g6qzrZqv5Ev+JEegtrA1DNV/FahI+FOJvlnRIkihoaee96Qz2z32E8B3gp1ztQxs5Bm2/nhWkotQu9ZikjtoK7UtSwcx3FaQXtH+4Vc47qV8FXbGH8QqWNgZWMVv1XpaFie07NYCvHwww/nLidhHg3FrFFXomJIpc5HN7744othCUpqk5J4jTc7imWdgPzXDAyhssVmPJxfBHOshFvX6RrHcZwikGtMMSLXYl2RmH379gX5ePXVV+cuM2iLXbumv4rfqnQk3LV/sF1n2w7NU2geNz4iEGUu3WM+nPlgHQHI3Abzc5qTYJ0kv3HXHIWUwXhW4XDfCpM4DqF4uId/m87UfDJ+eN4eUYg/xUU8RUc32nyk5mx41oYblxNUTW8K1sESTifwbnbv3h20msuiLUcPHToUbMdxnLmEeW21q7SX0vlAnshdcqQbdB5DfN6Atti1WzpX8VuZ/Au+EjxW5VEN4zPUwDAEJjXswNpJ3LiH4TfPafjDDoewjlNhMWSMO24KT8POhGNRHHbYGRg21tyyhqqVRjv0zJwgacI/cUPKH+CWGrqx+bBo7kXhkA784Ea6LWXTm4Iywl9cBoKw7T3Cw00Qr9Zzl6VdnBbluazpZujKcZzRQXLBtl+SM1X0PQgj1baD2q9UuxQ/V8VvVeZkKRxrqPmKfeqpp8KQBuaRRx4J995+++1gg3orDOuzTSPbUWYCK7iBhkPY/emZZ55phrVhw4bgfskllzSnA7SrEyMGlqIDCgiHoWfSpaFqDTnbXak4EIG8PPnkk830KC/tduYSes5COjlkIXuZzXhJB+UADz74YLBF2fR2AkPou3btCmliZOD1119vnu6kEY+HHnoo3EuNnnQLw19Z3SxtfB294zhlQC4gUzgUhxFI2jB25GNPDm21PCz0VLgzJJwa9qDRR2hZoSaBpCF+y2WXXZZfNYIQ1yEDQtsoxsRDG51g56D1su3+yAgSBEo3Rzem0NGH8V7dhE3ZIchTtEtvJ7ClKGvfsy/0cLAH12wqog4Ih6rwLjlMgvgpj/feey8sq+Mfpu6ojrpx42a4TBn4YBobG2vcfPPN4eME3bFhE+zQkXBfuHBhsDXHLCg0fWm/9dZbzQJLzT3rRRQJrTpDvpm/oWJo/rxbdPRhHUBw8y71ZUwHC+i8jY+Ph44Ngp53qlELOj38w3SjADJXKF9u3LgZLlMWdIaQS5gqumNliE9rFPrYtVTxW5WOhDtfcpBSjlqyZEmw4+Fvvj5TLwMzSFDovTi6MWbx4sX51elHw9i//vprsMuAUh/a/wxngc7StiMHdAqKFEB0sMbFF18c7FZoqL+sGYTRAsdx6gNTxZOTk+HjUpsn9QqNPP/www/BFjrj3o7OVvFblY6Eu46aLHvmNV977Y4IHBR6cXRjimXLlgU7HlLneb6Q6Rz1Er6y2d+7DKThrrvuCqc8WWFehc8//zzYF154YbBb4XPujuOcLrSiiKnGqamp8BXfyx001VYzj2/RqCanHYoqfiuTNY4dIc1sbGmNo/EnzXWr/ce1/GY9peCGjea01Z6WJnuRJra0zK3mNuAfd6tRLr+kx1IUB2HirrwAaYzjU74VFxqWcovTxY5buOOHcPkNRflQONJ2x5/cYm3KsuktotUmNjGUVRwmeYqfzypqoTY874H7juM4/YL2NG7LaJdoy2gTy6C2L5Ytlrgtp/1GIz8l26r4rULHwh1oyCXMMRQaBYQdLytAGOIe+xUqMJlUxsiw7lMgICElo3htuvQS4jgUv4Rw7DcVH8JUlYH7uEtYW39AGEoHzyhtqXBFXKakTc+JKuktgjTzbDt/hEt4cecC7PPqwKU6C7qXCsPpL/wPqN4Udczqjv1/GSTUeVf517Xza9vYQa0jYNtNtf2x/Gi1jFgfOdY/7473pvAslJXeLXW0VdlV8VuWwfpvcHqKhK4d8YihMhd1ABD8quxUyFQFVyei217oIMM/KmVUxvQDjWb1okHpFxLwgwT/OxrNQ6jUVbjDMNSRUWNO1rk79YS56kwgh/l0tP9TMC+PfkEKVkNwP6tHQbFQewsIFN3YqQ8FTGncjzKUNWWFgawxD9dZB2jWfg5zTdHeD4NE0fLYuoKCKrtEciwysEdFvOS3TgxDHRk1XLiPOAhkGhm0/8tuXVsGOgs7duwIx8kWdQ5GCQR53PkRrDJgg6dOYXUBxhkc6rT01RlOXLg7QQMeAdxLIcyXCJsXuSb7jFZuu68yBLy+6KvASoY6H1DkpKmyDNVxOsGFu+PUFKY1+NrXen6Gnu2BR3Seig4oEgqDHSMVRnwQURmIS2EwKkMYOsCIMO1eA4wCya9Nrz24Q/7xq42gSCe/tc8BYWhLY8JRmNwvWmKKOyNQ8kuYKb+Eaw9g4hnrT/cwuLNUSmHavMawLNbGnwqbsHbu3BmuV61aFfzZcrIoPyojDGlTuYh2+QF+E4/Cwk7FG/sjvKeffjq/Oxv82bLimV4uK3O6IPtacBxnjuFfr5UClZQdUWZEKRGDUiJusXJiUVg8yz0pOmpFBcaiuNopS0mpCiUwaRUXhVmkgKU82JUT5A038sB95VdKcrjJP3nBLVby5Fm5a8WG0oDimoXwSK/CxOZ3XIY8h8GdsFSeNu0W0kw4PKM0KL1xGqRkWRSWIM5UWm25Vs2P0sY7JA3xO+I5Gx7+eS72q7ypLshfHK/TH1y4O04foFFs1Qgi2GhgERgWCTw10FAUFm6xUJGAstCI4xY38jHyFwtWLTGyFIXJb9wlOARu5M0iv+qcCNzi/PIbd1suoPzSCQHu8xuBaJE/+7zC1LPqhMRpFwojvi93m4+icoghDZSLrQekXeVaNj8SxHRSLIRty13+4jJPvU/lQeUDhB+/G6c/+LC849QMhkWzRrmxYsWKMBdv0QmI2v63Fczzx7sQanvoVkPL7VAYQltodhMmMKSbosqBUPEOikrrF198Eex9+/YF+4Ybbgi2kL+U/oLOyJBeRJEeico6vr9+/fpg//bbb8GuAtuPUheYfmFag2F/Vp6gxwFl87N3795g2wOvgDInfO2yqfPFy+y3vnLlymCvXr06DMUzpUL4ddb6HyVcuDtOzThy5Eh+1T0IXOZfNY/NqX6jiI5Blq25bhmVSycCWLDqpNcgxLOv4XBY18aNG8NJjXZOu2x+jh8/HmzrB4OuBqizqLMh4k5lCjox2Vd7WOrKmRPLly9vqQ/hzC0u3B2nZpxzzjn5VXcg1DnWcu3atc39+icnJ/O7o8WCBQvyqxkOHDgQyiM2+iLuBATv6YCvYUYFEKTj4+NBkMaKcGXzk/KD0WhD0SllRTCqgTIfX//ULToLHAXt9B8X7o5TMxha5kstddjSnj17wj0dNCRifwyRopHNYT92KHaul2Dpy1LMdfyKT8PMa/KDOg4ePBjsXjI2NhbseHpCh4BoGLsKfF3r3UqQgg6YKpufdevWBbvd1AlD7BCvqDh27Fh+dRK+0uWPOktHgvRoNMDpLy7cHWeO0fxmq+FL9hxgidvWrVub/phzZegXgW2HTfma00ZE+GX51/z588M9hABuGJ7XvLCO4AVdtxO88lcksG2YfAnSCaGDofzytZmKX/mTLRRPyi87IqawS8DIL/FPTEw05+IRkAig7du3zxJgXFNuSisUpauIzZs3hzxv2rSpGQ7ClDQQp52LT5VZEXEdAAnrsvnhdDGljXoChElnAX+CThD+tm3bNisP+/fvD9fxu4/90SGlPjo14ITjOHMGmsVZ4xm0jDFWUzkGzWMtQcJwHWs7A2ESDn6yhr6pvYzGs+LSs9Jwxh1/cXrQsk6BP/nBSJtaWtkYhSnwo3QRPxrXNn6FkYqfe3LDKFyFh7FlR74JG819hcd93GLQPCce+cPmtzTLQSsAFI7NVyvwFz8bp6FVmcXgN85zHF6Z/EBR2uIVGfhTvZMfactjCBtYAmfrp+KNw3P6wzz+ZC/GcRzHcZwhwYflHcdxHGfIcOHuOI7jOEOGC3fHcRzHGTJcuDuO4zjOkOHC3XEcx3GGDBfujuM4jjNUNBr/B9wHepyFtl2VAAAAAElFTkSuQmCC\" width=\"503\" height=\"56\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHarvesting of TWW-grown cyanobacterial biomass for lipid analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh biomass of NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eSS\u003cem\u003e\u0026nbsp;\u003c/em\u003egrown in 100% TWW was harvested after 28 days of initial culturing by the filtration method. The harvested biomass was transferred in to aluminum trays and oven-dried at 55\u0026deg;C until they were fully dried. The dried biomass was made into a fine powder, measured and stored in the freezer (-4˚C) for lipid analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid extraction and quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipid extraction was done in a Soxhlet apparatus (Biobase\u0026reg;, China) using hexane as the extraction solvent. Two grams of dried TWW-grown biomass of NE and SS was taken and wrapped in a Whatman No. 1 qualitative filter paper. It was refluxed with 50 ml of hexane for six hours with a water condenser. The hexane layer containing the extracted lipids was collected and vacuum-dried in a rotary evaporator (Heidolph\u0026trade;, Germany) at 37 ˚C to evaporate the solvent. The dried lipid was weighed and the total lipid content was calculated gravimetrically on w/w % of dry biomass by taking the difference in the pre- and final weights of the vial. For each treatment, three replicates were maintained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"616\" height=\"61\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFatty acid profile analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFatty acid methyl esters (FAMEs)/ biodiesel were prepared from extracted lipids following an acid-base transesterification reaction as follows; 200 \u0026micro;l of lipids were reacted with 2 ml of methanol containing 2% (w/v) NaOH for 15 min at 65 \u0026ordm;C in a water bath. Then, 2 ml of methanol containing 5% (v/v) H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added to the screw-capped glass tube and heated for another 15 min at 65 \u0026ordm;C. Esterified fatty acids were extracted into 1 ml of hexane by centrifugation (1500 rpm for 10 mins) (Javed et al., 2022). FAMEs were analyzed using a gas chromatograph (Agilent 7890B, CA, USA) equipped with a flame ionization detector (FID) in a capillary column (100 m\u0026times;0.25 mm\u0026times;0.2 \u0026mu;m) (Agilent J \u0026amp; W CP-Sil 88 for FAME, CA, USA). Ultra-high purity nitrogen gas (99.9995%) was used as the carrier gas. Initial column temperature was set at 100 \u0026deg;C, which was progressively raised to 180 \u0026deg;C at 8 \u0026deg;C/min, and then to 230 \u0026deg;C at 1 \u0026deg;C/min. The injector was retained at 260 \u0026deg;C with an injection volume of 1 \u0026mu;L in a splitless mode. The peaks were identified by comparing the retention times with those of a mixture of external standard methyl esters (Supelco\u0026reg; 37 FAME Mix, Sigma Aldrich, St. Louis, MO, USA). The results were expressed as the relative percentage of total peak area of FAMEs considered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were presented as mean \u0026plusmn; standard deviation. The statistical analysis of the data was performed using analysis of variance (ANOVA) and when significant effects were detected, the groups were compared using a post-hoc Tukey\u0026rsquo;s HSD test. The level of significance used for all statistical tests is 5% (p \u0026lt; 0.05). The statistical program used was Minitab version 19.2.\u003c/p\u003e"},{"header":"Results \u0026 Discussion","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of textile wastewater\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pH, electric conductivity (EC), salinity and total dissolved solids (TDS) were recorded as 9.93, 704\u0026nbsp;mS, 350 mg/L and 470 mg/L, respectively. The initial characteristics of the TWW revealed a high COD value of 3046 mg/L, along with higher concentrations of phosphates (168 mg/L), ammoniacal nitrogen (70 mg/L), and nitrates (200 mg/L) (Table 1). These findings highlighted the necessity for adequate treatment of TWW before discharge into the environment.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCyanobacterial growth in textile wastewater\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth patterns of NE and SS in TWW were assessed by measuring optical density (OD) at 680 nm (Fig. 1), a standard wavelength for monitoring cyanobacterial growth and chlorophyll content. Both strains exhibited a gradual increase in OD values over the cultivation period, indicating successful adaptation and proliferation in the wastewater environment. Among the two, NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eshowed the most promising growth, achieving a growth rate of 90.82% \u0026plusmn; 0.09, consistently displaying a higher OD increase compared to SS, which recorded a growth rate of 83.57% \u0026plusmn; 0.06.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe observed growth trends highlighted the adaptive capabilities of cyanobacteria in TWW, demonstrating their ability to utilize TWW as a nutrient source while tolerating its toxic components. Cyanobacteria are known for their resilience to environmental stressors and efficient uptake of inorganic nutrients (e.g., ammoniacal nitrogen, nitrates and phosphates) from industrial effluents, promoting biomass accumulation (Cai et al., 2013). The higher growth rate of NE (a heterocystous filamentous cyanobacterium)\u003cem\u003e\u0026nbsp;\u003c/em\u003emay be attributed to its nitrogen-fixing ability, allowing it to thrive even in nitrogen-limited conditions commonly found in TWW. Conversely, SS is a non-heterocystous cyanobacterium that relies on available nitrogen sources for growth, which may explain its relatively lower but still significant growth rate. Inorganic phosphates presented in the TWW also could have contributed to the notable algal growth, playing a key role in their metabolism (Mouga et al., 2024). These findings suggest that TWW could serve as a low-cost, sustainable cultivation medium for cyanobacterial biomass production, reducing the need for synthetic growth media while simultaneously supporting bioremediation applications. Further optimization of cultivation parameters, such as light intensity, aeration, and supplementation with additional nutrients, could enhance the growth efficiency of these strains in industrial-scale applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecolorization of textile wastewater\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDecolorization of TWW by NE and SS were evaluated by measuring OD at 775 nm. Significant color removal of TWW was observed from NE and SS with 79.03% \u0026plusmn; 0.09 and 82.67% \u0026plusmn; 0.09, respectively (Fig. 2). These results indicated that both strains possess strong biosorption and biodegradation capabilities, contributing to dye removal from TWW. The observed decolorization can be attributed to several mechanisms, including biosorption (cell surface adsorption), bioaccumulation (active transport of dye molecules in to the cell), extracellular polymeric substance (EPS) secretion, and enzymatic degradation (biodegradation). The negatively charged cell walls of cyanobacteria facilitate electrostatic interactions with dye molecules, allowing passive dye adsorption, while intracellular metabolic processes and oxidative enzymes (e.g., azoreductases, laccases, peroxidases) further contribute to biodegradation (Dudeja et al., 2025; Mishra \u0026amp; Maiti, 2019; Sudarshan et al., 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical Oxygen Demand (COD) removal efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NE and SS significantly reduced the initial COD value of 3042 mg/L to 107 mg/L and 210 mg/L, achieving COD removal efficiencies of 96.49% and 93.11%, respectively. This directly implied their ability to successfully consume organic matter present in TWW suggesting their capability to bioremediate TWW with high COD levels. A high COD level indicates a complex mixture of organic pollutants, which may require extended treatment time to remediate (Nguyen et al., 2022). Since COD is an indirect measure of organic compound concentration, assessing its reduction is crucial before TWW is discharged into the environment. According to Selvaraj \u0026amp; Arivazhagan, (2024), there is a strong correlation between high biomass productivity and COD removal efficiency since cyanobacteria utilize organic carbon as an energy source and a nutrient substrate for their growth. This ultimately improves the wastewater quality into permissible levels and it was evident in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effective COD utilization by NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand SS can be attributed to factors such as species-specific metabolic activity, growth conditions, and nutrient availability in TWW. Additionally, oxygen generation by these photosynthetic prokaryotes enhances the biological degradation of organic matter, further contributing to COD reduction in TWW (Selvaraj \u0026amp; Arivazhagan, 2024). In a similar study, Javed et al., (2022) observed a COD removal of 59% by \u003cem\u003eChlorella vulgaris\u003c/em\u003e on unsterilized, 100% TWW, which was lower than the COD removal efficiencies observed in this study using \u003cem\u003eNostoc\u003c/em\u003e sp. and \u003cem\u003eSpirulina\u003c/em\u003e sp. Brar et al., (2019) observed a 50% reduction of COD by \u003cem\u003eAnaebaena ambigua\u003c/em\u003e in sterilized, 75% TWW. Raza et al., (2024) observed a COD reduction of 91.5% by a consortium of \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e sp.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutrient removal efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant reduction in the concentration of ammoniacal nitrogen, nitrate and phosphate present in the TWW after the phycoremediation experiment.\u0026nbsp;NE achieved a 76.28% ammoniacal nitrogen removal, while SS exhibited a higher removal of 96.86%. The nitrate reduction was recorded as 69% and 73% with NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand SS, respectively. Meanwhile, the phosphate removal was detected as 81.14% with NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand 33.33% with SS. Overall, SS demonstrated a superior nitrogen removal efficiency, whereas NE\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas more efficient in phosphate removal. All these findings highlighted the potential of selected cyanobacterial species for nutrient removal from TWW. These reductions in nutrient levels corresponded with an increase in algal biomass, demonstrating a direct relationship between nutrient uptake and growth. However, some studies indicated that phosphorus uptake by cyanobacteria may not always be efficient, depending on species and environmental conditions (Tsolcha et al., 2017), which is also evident by SS\u003cem\u003e\u0026nbsp;\u003c/em\u003ein this study.\u003c/p\u003e\n\u003cp\u003ePhosphorus (P) and Nitrogen (N) are key pollutants present in wastewater, posing significant threats to aquatic ecosystems, leading to eutrophication (Preisner et al., 2021). Cyanobacteria play a crucial role in nutrient removal from wastewater through N assimilation and phosphorylation. These two processes facilitate the uptake of N and P, which are vital for algal metabolism, growth, and biomass production. They assimilate N by converting inorganic N sources: nitrate (NO₃⁻), nitrite (NO₂⁻), and ammonium (NH₄⁺) into organic forms required for the synthesis of peptides, proteins, enzymes, chlorophylls, and genetic materials (RNA and DNA) via enzymatic reduction (Inabe et al., 2021; Raza et al., 2024). Among the different N sources, ammonium (NH₄⁺) is preferred by cyanobacteria due to its lower energy requirement for assimilation. Wastewater rich in ammonium, therefore, serves as a promising medium for cyanobacteria cultivation (Nguyen et al., 2022; Znad et al., 2018). Phosphorus is another essential macronutrient required for the production of lipids, proteins, nucleic acids, and ATP (adenosine triphosphate). Phosphate assimilation by cyanobacteria through phosphorylation is the primary mechanism for phosphate removal in wastewater (Znad et al., 2018). Inorganic phosphorus exists primarily in the forms of H₂PO₄⁻ (phosphoric acid) and HPO₄\u0026sup2;⁻ (hydrogen phosphate). These forms are absorbed by cyanobacteria through phosphorylation, where phosphate is incorporated into intracellular organic compounds via specialized phosphate transporters in the plasma membrane (Burford et al., 2023; Markou et al., 2014)\u003c/p\u003e\n\u003cp\u003eVarious environmental factors affect the nutrient uptake rates in different cyanobacterial species, including initial nutrient concentration, light intensity, airflow rate, extracellular pH, temperature, and inoculum size (Tsolcha et al., 2017). Due to their diverse physiological and morphological traits, different species of cyanobacteria demonstrate varied effectiveness in removing N and P from wastewater effluents. Hence, the selection of an appropriate algal strain is crucial for optimizing nutrient removal efficiency. Based on a study by Brar et al., (2019), a nitrate removal of 52.95% and phosphate removal of 63.05% were achieved on sterilized, 75% TWW by \u003cem\u003eAnaebaena ambigua. Raza et al., (2024)\u003c/em\u003eobserved increased removal efficiencies by a \u003cem\u003eChlorella vulgaris-Staphylococcus\u003c/em\u003e consortium achieving NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e (58.57%), and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e (89.19%), compared to algae only treatment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003ePhysicochemical characteristics of textile wastewater before and after treatment with \u003cem\u003eNostoc ellipsosporum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eSpirulina\u003c/em\u003e \u003cem\u003esubsalsa\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 278px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInitial assessment of TWW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTWW treated with\u003cem\u003e\u0026nbsp;Nostoc ellipsosporum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTWW treated with\u003cem\u003e\u0026nbsp;Spirulina\u003c/em\u003e \u003cem\u003esubsalsa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristic of TWW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBefore treatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter treatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRemoval efficiency (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter treatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRemoval efficiency (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e9.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e8.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e8.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eElectric conductivity (EC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026mu;S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e704\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e1677\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSalinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eTotal dissolved solids (TDS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e20.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e390\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e17.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eChemical oxygen demand (COD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e3046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e96.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e93.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eTotal phosphate (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e31.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e81.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e33.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAmmoniacal nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e16.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e76.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e96.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eNitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of cyanobacteria-treated textile wastewater on seed germination and seedling growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of tap water (positive control), untreated TWW (negative control) and treated TWW by NE (NE-TWW) and SS (SS-TWW) on seed germination and seedling growth are depicted in Table 2. Seeds in the positive control, NE-TWW and SS-TWW treatments exhibited a 100% germination rate, whereas the negative control had a significantly lower germination rate of 25%. The initial physicochemical analysis revealed that TWW was toxic in nature due to the excess pollutant concentrations as shown in the Table 1. It was further confirmed by having an inhibitory effect on seed germination and seedling growth. After phycoremediation, toxicity of TWW was significantly reduced and showed improved seed germination indicating their pollutant removal capability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA seed was deemed to have successfully germinated if its root system were visible and measurable, with a minimum length of 1 mm. Seeds that did not germinate were assigned a root length of zero (Rekik et al., 2017). In this study, shoot length was highest in the positive control (8.12 cm), followed by NE-TWW (6.88 cm) and SS-TWW (6.54 cm), while the negative control exhibited the shortest shoot length (1.99 cm). Similarly, root length followed the same trend, with the positive control (6.44 cm) having the longest roots, followed by NE-TWW (4.97 cm) and SS-TWW (4.24 cm), whereas the negative control had the shortest root length (1.02 cm). These findings are in accordance with a similar study conducted by Selvaraj \u0026amp; Arivazhagan, (2024), which had maximum shoot and root lengths of approximately 8.5 cm and 5 cm, after 05 days irrigated with TWW treated with \u003cem\u003eSpirulina platensis\u003c/em\u003e. Elakbawy et al., (2022)reported a shoot length of 5.69 cm on tomato seedlings when testing the effect of \u003cem\u003eNostoc muscorum\u003c/em\u003e crude extracts on treated sewage wastewater.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCyanobacteria release secondary metabolites to the medium during their growth, including plant growth hormones, micronutrients, macronutrients, and vitamins (Elakbawy et al., 2022). The release of these beneficial compounds by cyanobacteria also could be one of the reasons for the enhanced shoot and root development observed in the present study. These results suggest that the phycoremediated TWW enhances the germination process by providing additional nutrients to the medium, highlighting the potential use of phycoremediated TWW for irrigation. The reason for reduced seed growth in untreated TWW could be due to the excess nutrient concentrations, high levels of COD, and heavy metals which retard seedling growth by affecting water absorption and other metabolic activities (Dhaouefi et al., 2019). Different crop plant species also differ widely in response to different concentrations of wastewater with respect to seed germination, seedling growth and productivity (Fendri et al., 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003ePhytotoxicity results of treatments showing germination (%), shoot length and root length\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTap water\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTWW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNE-TWW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSS-TWW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGermination (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eShoot length (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e8.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e1.99\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e6.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e6.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoot length (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e6.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e1.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e4.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e4.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTWW: Textile wastewater, NE-TWW: \u003cem\u003eNostoc\u003c/em\u003e-treated TWW, SS-TWW: \u003cem\u003eSpirulina\u003c/em\u003e-treated TWW\u003c/p\u003e\n\u003cp\u003eMeans followed by different superscript letters within a row are significantly different at p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal lipid content of textile wastewater-grown cyanobacterial biomass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh lipid content in cyanobacterial cells is a key factor determining their potential for biodiesel production (Hawrot-Paw et al., 2021; Nagappan et al., 2020). The total lipid contents of the TWW-grown cyanobacterial strains are shown in Fig. 3. It is worth mentioning that, NE\u003cem\u003e\u0026nbsp;\u003c/em\u003eand SS\u003cem\u003e\u0026nbsp;\u003c/em\u003edemonstrated significant lipid yields of 21.5% and 25%, respectively, compared to several cyanobacterial strains used in TWW bioremediation (Mathimani et al., 2024). To the best of our knowledge, this is the first report that the lipid content and lipid profile of NE and SS\u003cem\u003e\u0026nbsp;\u003c/em\u003egrown in unsterilized and undiluted TWW have been analysed. These results suggest that both strains can thrive in unsterilized-raw TWW, utilizing it as a favorable growth medium while achieving enhanced lipid production. The variation in lipid content among two species could be a result from differences in their morphology, biochemical composition, and native environmental adaptations. Moreover, lipid profile and productivity are also influenced by multiple factors, including cultivation conditions, biomass harvesting, cell disruption methods, strain selection, solvent polarity, and extraction techniques (Fazal et al., 2018; Javed et al., 2022).\u003c/p\u003e\n\u003cp\u003eHigher lipid accumulation in algae under wastewater conditions is attributed to nutrient limitation, which enhance lipid synthesis (Feng et al., 2011; Mahmood et al., 2023). Specifically, the nitrogen (N) and phosphorus (P) levels in wastewater may be lower than those in standard growth media (i.e., BG-11, Zarrouk), inducing nutrient starvation that triggers lipid accumulation (Purba et al., 2022). Mostafa et al., (2012) reported lipid contents of 15.5% and 7.3% for \u003cem\u003eNostoc humifusum\u003c/em\u003e and \u003cem\u003eSpirulina platensis\u003c/em\u003e, respectively, when cultivated in domestic wastewater for biodiesel production. Hossain et al., (2018) reported a lipid content of 15.43% in \u003cem\u003eNostoc\u003c/em\u003e sp. cultured in half-strength BG-11 medium, while Nagappan et al., (2020) observed a lipid content of 15.7% in \u003cem\u003eNostoc\u003c/em\u003e sp. MCC41 when grown in BG-11 medium. Ambrozova et al., (2014) reported a lipid content of 13.41% for \u003cem\u003eSpirulina platensis\u003c/em\u003e cultivated in BG-11, whereas Haberle et al., (2020) obtained a lipid content of 15.3% for \u003cem\u003eSpirulina subsalsa\u0026nbsp;\u003c/em\u003egrown in oil refinery wastewater. These findings highlighted the notable lipid yields achieved in TWW-grown NS and SS in this study, reinforcing their potential as viable feedstocks for biodiesel production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFatty acid methyl ester (FAME) analysis for biodiesel production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of FAME profile is crucial to determine the biodiesel quality of cyanobacteria (Mathimani et al., 2024). Therefore, the fatty acid composition of TWW-grown NE and SS\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas analyzed using GC-FID. In algal FAME profiles, fatty acids with carbon chain lengths of C16\u0026ndash;C18 are the most commonly identified, as also confirmed by the results of the present study. The key FAMEs that determine biodiesel quality include palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and low concentrations of linoleic acid (C18:2), and linolenic acid (C18:3) (Hawrot-Paw et al., 2021). According to Knothe, (2005), two critical factors influencing biodiesel quality are oxidative stability and low-temperature performance.\u003c/p\u003e\n\u003cp\u003eIn the present study, both cyanobacterial strains exhibited a similar fatty acid composition, though slight variations were observed in the percentages of each fatty acid. The key FAMEs identified in both cyanobacterial strains are, palmitic acid (C16:0), oleic acid (C18:1), stearic acid (C18:0), lauric acid (C12:0), and myristic acid (C14:0) (Table 3). Notably, palmitic acid and oleic acid together accounted for more than 70% of the dominant FAMEs recorded in both species. Similarly, \u003cem\u003ePhormidium lucidum\u003c/em\u003e grown in oil refinery wastewater was investigated for biodiesel production and was found to have palmitic and oleic acids as the predominant fatty acids, comprising 64% of the total FAMEs (Haberle et al., 2020), which is consistent with our findings.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCyanobacterial lipids are rich in both saturated and unsaturated fatty acids. Unsaturated fatty acids (UFAs) enhance biodiesel\u0026rsquo;s cold flow and cloud point, preventing fuel solidification in cold weather. However, UFAs are more prone to oxidation and rancidification unlike saturated fatty acids (SFAs). In contrast, SFAs contribute to crystal formation at low temperatures, leading to poor cold flow properties (Sadvakasova et al., 2021; Sinha et al., 2016). Since UFAs improve low-temperature performance, while SFAs enhance oxidative stability, a well-balanced FAME composition is essential for optimal biodiesel quality, specifically with low levels of UFAs. Among UFAs, monounsaturated fatty acids (MUFAs) are ideal for biodiesel production, whereas polyunsaturated fatty acids (PUFAs) are less favorable. Oleic acid (C18:1), a MUFA, is considered optimal due to its balanced oxidative stability and low-temperature performance (Feng et al., 2011; Knothe, 2005). High PUFA content can increase nitrogen oxide emissions, but a low PUFA concentration positively affects biodiesel\u0026rsquo;s flow properties, particularly at low temperatures (Hawrot-Paw et al., 2021). In this study, palmitic acid (C16:0) was the most abundant fatty acid, followed by oleic acid (C18:1) in both strains. NE contained 45.92% (C16:0) and 24.77% (C18:1), while SS\u003cem\u003e\u0026nbsp;\u003c/em\u003ehad 45.88% (C16:0) and 28.13% (C18:1). As shown in Fig. 3, the SFA and MUFA percentages in both strains was optimal (Fig. 3), suggesting their suitability for third-generation biodiesel production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Most dominant fatty acid methyl esters present in the textile wastewater-grown cyanobacterial biomass\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of fatty acid methyl ester\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbon number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFatty acid percentage (%) in NE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFatty acid percentage (%) in SS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003ePalmitic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC16:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e45.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e45.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eOleic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC18:1 (cis (n9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e24.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e28.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eStearic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC18:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e8.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e5.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eCis-10-pentadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC15:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e8.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eCis-10-heptadecanoic acid\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC17:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e8.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eLauric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC12:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e6.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e6.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eMyristic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003eC14:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e5.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e5.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated the dual potential of NE and SS in TWW bioremediation and biodiesel production. Both cyanobacterial species exhibited significant growth in undiluted and unsterilized TWW, effectively removing pollutants while achieving high decolorization efficiencies. NE and SS exhibited notable COD removal efficiencies of 96.48% and 93.11%, respectively. SS showed superior ammoniacal nitrogen removal (96.86%), whereas NE exhibited better phosphate removal (81.14%) and nitrate removal (73%). The treated wastewater significantly improved seed germination and seedling growth, highlighting its potential application for irrigation. Furthermore, the TWW-grown cyanobacterial biomass demonstrated high lipid accumulation, with SS yielding 25% total lipids and NE yielding 21.5%. Fatty acid profiling confirmed the presence of key biodiesel precursors, such as palmitic acid (C16:0) and oleic acid (C18:1), indicating their suitability for sustainable biofuel production. This integrated approach presents a cost-effective and environmentally friendly alternative for TWW treatment, while simultaneously producing biodiesel from wastewater-grown biomass.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e The authors declare that no funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by S.M.D.C. Bandara. The first draft of the manuscript was written by S.M.D.C. Bandara and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAmbrozova, J. V., Misurcova, L., Vicha, R., Machu, L., Samek, D., Baron, M., Mlcek, J., Sochor, J., \u0026amp; Jurikova, T. (2014). Influence of extractive solvents on lipid and fatty acids content of edible freshwater algal and seaweed products, the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(2), 2344\u0026ndash;2360. https://doi.org/10.3390/molecules19022344\u003c/li\u003e\n \u003cli\u003eAPHA, 2012. Standard Methods for the Examination of Water and Wastewater, twenty- second ed. American Public Health Association, Washington, DC.\u003c/li\u003e\n \u003cli\u003eBharagava, R. N., \u0026amp; Bhimrao, B. (2018). \u003cem\u003eTextile Industry Wastewater: Environmental and Health Hazards and Treatment Approaches\u003c/em\u003e. https://www.researchgate.net/publication/328701616\u003c/li\u003e\n \u003cli\u003eBrar, A., Kumar, M., Vivekanand, V., \u0026amp; Pareek, N. (2019). Phycoremediation of textile effluent-contaminated water bodies employing microalgae: nutrient sequestration and biomass production studies. \u003cem\u003eInternational Journal of Environmental Science and Technology\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(12), 7757\u0026ndash;7768. https://doi.org/10.1007/s13762-018-2133-9\u003c/li\u003e\n \u003cli\u003eBurford, M. A., Willis, A., Xiao, M., Prentice, M. J., \u0026amp; Hamilton, D. P. (2023). Understanding the relationship between nutrient availability and freshwater cyanobacterial growth and abundance. \u003cem\u003eInland Waters\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(2), 143\u0026ndash;152. https://doi.org/10.1080/20442041.2023.2204050\u003c/li\u003e\n \u003cli\u003eCai, T., Park, S. Y., \u0026amp; Li, Y. (2013). Nutrient recovery from wastewater streams by microalgae: Status and prospects. In \u003cem\u003eRenewable and Sustainable Energy Reviews\u003c/em\u003e (Vol. 19, pp. 360\u0026ndash;369). https://doi.org/10.1016/j.rser.2012.11.030\u003c/li\u003e\n \u003cli\u003eCuellar-Bermudez, S. P., Aleman-Nava, G. S., Chandra, R., Garcia-Perez, J. S., Contreras-Angulo, J. R., Markou, G., Muylaert, K., Rittmann, B. E., \u0026amp; Parra-Saldivar, R. (2017). Nutrients utilization and contaminants removal. A review of two approaches of algae and cyanobacteria in wastewater. \u003cem\u003eAlgal Research\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e, 438\u0026ndash;449. https://doi.org/10.1016/j.algal.2016.08.018\u003c/li\u003e\n \u003cli\u003eDhaouefi, Z., Toledo-Cervantes, A., Ghedira, K., Chekir-Ghedira, L., \u0026amp; Mu\u0026ntilde;oz, R. (2019). Decolorization and phytotoxicity reduction in an innovative anaerobic/aerobic photobioreactor treating textile wastewater. \u003cem\u003eChemosphere\u003c/em\u003e, \u003cem\u003e234\u003c/em\u003e, 356\u0026ndash;364. https://doi.org/10.1016/j.chemosphere.2019.06.106\u003c/li\u003e\n \u003cli\u003eDudeja, C., Masroor, S., Mishra, V., Kumar, K., Sansar, S., Yadav, P., Chaturvedi, N., Singh, R., \u0026amp; Kumar, A. (2025). Cyanobacteria-based bioremediation of environmental contaminants: advances and computational insights. \u003cem\u003eDiscover Agriculture\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(1), 42. https://doi.org/10.1007/s44279-025-00193-9\u003c/li\u003e\n \u003cli\u003eElakbawy, W. M., Shanab, S. M. M., \u0026amp; Shalaby, E. A. (2022). Enhancement of plant growth regulators production from microalgae cultivated in treated sewage wastewater (TSW). \u003cem\u003eBMC Plant Biology\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(1). https://doi.org/10.1186/s12870-022-03764-w\u003c/li\u003e\n \u003cli\u003eEl-Sheekh, M., El-Dalatony, M. M., Thakur, N., Zheng, Y., \u0026amp; Salama, E. S. (2022). Role of microalgae and cyanobacteria in wastewater treatment: genetic engineering and omics approaches. In \u003cem\u003eInternational Journal of Environmental Science and Technology\u003c/em\u003e (Vol. 19, Issue 3, pp. 2173\u0026ndash;2194). Springer Science and Business Media Deutschland GmbH. https://doi.org/10.1007/s13762-021-03270-w\u003c/li\u003e\n \u003cli\u003eEl-Sheekh, M. M., Galal, H. R., Mousa, A. S. H., \u0026amp; Farghl, A. A. M. (2023). Coupling wastewater treatment, biomass, lipids, and biodiesel production of some green microalgae. \u003cem\u003eEnvironmental Science and Pollution Research\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(12), 35492\u0026ndash;35504. https://doi.org/10.1007/s11356-023-25628-y\u003c/li\u003e\n \u003cli\u003eFazal, T., Mushtaq, A., Rehman, F., Ullah Khan, A., Rashid, N., Farooq, W., Rehman, M. S. U., \u0026amp; Xu, J. (2018). Bioremediation of textile wastewater and successive biodiesel production using microalgae. In \u003cem\u003eRenewable and Sustainable Energy Reviews\u003c/em\u003e (Vol. 82, pp. 3107\u0026ndash;3126). Elsevier Ltd. https://doi.org/10.1016/j.rser.2017.10.029\u003c/li\u003e\n \u003cli\u003eFendri, I., Ben Saad, R., Khemakhem, B., Ben Halima, N., Gdoura, R., \u0026amp; Abdelkafi, S. (2013). Effect of treated and untreated domestic wastewater on seed germination, seedling growth and amylase and lipase activities in Avena sativa L. \u003cem\u003eJournal of the Science of Food and Agriculture\u003c/em\u003e, \u003cem\u003e93\u003c/em\u003e(7), 1568\u0026ndash;1574. https://doi.org/10.1002/jsfa.5923\u003c/li\u003e\n \u003cli\u003eFeng, D., Chen, Z., Xue, S., \u0026amp; Zhang, W. (2011). Increased lipid production of the marine oleaginous microalgae Isochrysis zhangjiangensis (Chrysophyta) by nitrogen supplement. \u003cem\u003eBioresource Technology\u003c/em\u003e, \u003cem\u003e102\u003c/em\u003e(12), 6710\u0026ndash;6716. https://doi.org/10.1016/j.biortech.2011.04.006\u003c/li\u003e\n \u003cli\u003eHaberle, I., Hrustić, E., Petrić, I., Priti\u0026scaron;anac, E., \u0026Scaron;ilović, T., Magić, L., Geček, S., Budi\u0026scaron;a, A., \u0026amp; Blažina, M. (2020). Adriatic cyanobacteria potential for cogeneration biofuel production with oil refinery wastewater remediation. \u003cem\u003eAlgal Research\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e. https://doi.org/10.1016/j.algal.2020.101978\u003c/li\u003e\n \u003cli\u003eHasan, M. N., Altaf, M. M., Khan, N. A., Khan, A. H., Khan, A. A., Ahmed, S., Kumar, P. S., Naushad, M., Rajapaksha, A. U., Iqbal, J., Tirth, V., \u0026amp; Islam, S. (2021). Recent technologies for nutrient removal and recovery from wastewaters: A review. \u003cem\u003eChemosphere\u003c/em\u003e, \u003cem\u003e277\u003c/em\u003e. https://doi.org/10.1016/j.chemosphere.2021.130328\u003c/li\u003e\n \u003cli\u003eHawrot-Paw, M., Ratomski, P., Koniuszy, A., Golimowski, W., Teleszko, M., \u0026amp; Grygier, A. (2021). Fatty acid profile of microalgal oils as a criterion for selection of the best feedstock for biodiesel production. \u003cem\u003eEnergies\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(21). https://doi.org/10.3390/en14217334\u003c/li\u003e\n \u003cli\u003eHossain, T., Miah, A. B., Mahmud, S. A., \u0026amp; Mahin, A. Al. (2018). Enhanced Bioethanol Production from Potato Peel Waste Via Consolidated Bioprocessing with Statistically Optimized Medium. \u003cem\u003eApplied Biochemistry and Biotechnology\u003c/em\u003e, \u003cem\u003e186\u003c/em\u003e(2), 425\u0026ndash;442. https://doi.org/10.1007/s12010-018-2747-x\u003c/li\u003e\n \u003cli\u003eInabe, K., Miichi, A., Matsuda, M., Yoshida, T., Kato, Y., Hidese, R., Kondo, A., \u0026amp; Hasunuma, T. (2021). Nitrogen availability affects the metabolic profile in cyanobacteria. \u003cem\u003eMetabolites\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(12). https://doi.org/10.3390/metabo11120867\u003c/li\u003e\n \u003cli\u003eJaved, F., Rehman, F., Khan, A. U., Fazal, T., Hafeez, A., \u0026amp; Rashid, N. (2022). Real textile industrial wastewater treatment and biodiesel production using microalgae. \u003cem\u003eBiomass and Bioenergy\u003c/em\u003e, \u003cem\u003e165\u003c/em\u003e. https://doi.org/10.1016/j.biombioe.2022.106559\u003c/li\u003e\n \u003cli\u003eKhan, S., \u0026amp; Malik, A. (2014). Environmental and health effects of textile industry wastewater. In \u003cem\u003eEnvironmental Deterioration and Human Health: Natural and Anthropogenic Determinants\u003c/em\u003e (Vol. 9789400778900, pp. 55\u0026ndash;71). Springer Netherlands. https://doi.org/10.1007/978-94-007-7890-0_4\u003c/li\u003e\n \u003cli\u003eKhatri, J., Nidheesh, P. V., Anantha Singh, T. S., \u0026amp; Suresh Kumar, M. (2018). Advanced oxidation processes based on zero-valent aluminium for treating textile wastewater. \u003cem\u003eChemical Engineering Journal\u003c/em\u003e, \u003cem\u003e348\u003c/em\u003e, 67\u0026ndash;73. https://doi.org/10.1016/j.cej.2018.04.074\u003c/li\u003e\n \u003cli\u003eKishor, R., Purchase, D., Ferreira, L. F. R., Mulla, S. I., Bilal, M., \u0026amp; Bharagava, R. N. (2020). Environmental and Health Hazards of Textile Industry Wastewater Pollutants and Its Treatment Approaches. In \u003cem\u003eHandbook of Environmental Materials Management\u003c/em\u003e (pp. 1\u0026ndash;24). Springer International Publishing. https://doi.org/10.1007/978-3-319-58538-3_230-1\u003c/li\u003e\n \u003cli\u003eKnothe, G. (2005). Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. \u003cem\u003eFuel Processing Technology\u003c/em\u003e, \u003cem\u003e86\u003c/em\u003e(10), 1059\u0026ndash;1070. https://doi.org/10.1016/j.fuproc.2004.11.002\u003c/li\u003e\n \u003cli\u003eMahmood, T., Hussain, N., Shahbaz, A., Mulla, S. I., Iqbal, H. M. N., \u0026amp; Bilal, M. (2023). Sustainable production of biofuels from the algae-derived biomass. \u003cem\u003eBioprocess and Biosystems Engineering\u003c/em\u003e, \u003cem\u003e46\u003c/em\u003e(8), 1077\u0026ndash;1097. https://doi.org/10.1007/s00449-022-02796-8\u003c/li\u003e\n \u003cli\u003eMarkou, G., Vandamme, D., \u0026amp; Muylaert, K. (2014). Microalgal and cyanobacterial cultivation: The supply of nutrients. In \u003cem\u003eWater Research\u003c/em\u003e (Vol. 65, pp. 186\u0026ndash;202). Elsevier Ltd. https://doi.org/10.1016/j.watres.2014.07.025\u003c/li\u003e\n \u003cli\u003eMathimani, T., Alshiekheid, M. A., Sabour, A., Le, T. H. T., \u0026amp; Xia, C. (2024). Appraising the phycoremediation potential of cyanobacterial strains Phormidium and Oscillatoria for nutrient removal from textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass. \u003cem\u003eEnvironmental Research\u003c/em\u003e, \u003cem\u003e241\u003c/em\u003e. https://doi.org/10.1016/j.envres.2023.117628\u003c/li\u003e\n \u003cli\u003eMishra, S., \u0026amp; Maiti, A. (2019). Applicability of enzymes produced from different biotic species for biodegradation of textile dyes. In \u003cem\u003eClean Technologies and Environmental Policy\u003c/em\u003e (Vol. 21, Issue 4, pp. 763\u0026ndash;781). Springer Verlag. https://doi.org/10.1007/s10098-019-01681-5\u003c/li\u003e\n \u003cli\u003eMona, S., Kumar, V., Deepak, B., \u0026amp; Kaushik, A. (2020). Cyanobacteria: The Eco-Friendly Tool for the Treatment of Industrial Wastewaters. In \u003cem\u003eBioremediation of Industrial Waste for Environmental Safety\u003c/em\u003e (pp. 389\u0026ndash;413). Springer Singapore. https://doi.org/10.1007/978-981-13-3426-9_16\u003c/li\u003e\n \u003cli\u003eMostafa, S. S. M., Shalaby, E. A., \u0026amp; Mahmoud, G. I. (n.d.). Cultivating Microalgae in Domestic Wastewater for Biodiesel Production. \u003cem\u003eNot Sci Biol\u003c/em\u003e, \u003cem\u003e2012\u003c/em\u003e(1), 56\u0026ndash;65. www.notulaebiologicae.ro\u003c/li\u003e\n \u003cli\u003eMouga, T., Pereira, J., Moreira, V., \u0026amp; Afonso, C. (2024). Unveiling the Cultivation of Nostoc sp. under Controlled Laboratory Conditions. \u003cem\u003eBiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(5). https://doi.org/10.3390/biology13050306\u003c/li\u003e\n \u003cli\u003eNagappan, S., Bhosale, R., Duc Nguyen, D., Pugazendhi, A., Tsai, P. C., Chang, S. W., Ponnusamy, V. K., \u0026amp; Kumar, G. (2020). Nitrogen-fixing cyanobacteria as a potential resource for efficient biodiesel production. \u003cem\u003eFuel\u003c/em\u003e, \u003cem\u003e279\u003c/em\u003e. https://doi.org/10.1016/j.fuel.2020.118440\u003c/li\u003e\n \u003cli\u003eNguyen, L. N., Aditya, L., Vu, H. P., Johir, A. H., Bennar, L., Ralph, P., Hoang, N. B., Zdarta, J., \u0026amp; Nghiem, L. D. (2022). Nutrient Removal by Algae-Based Wastewater Treatment. \u003cem\u003eCurrent Pollution Reports\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(4), 369\u0026ndash;383. https://doi.org/10.1007/s40726-022-00230-x\u003c/li\u003e\n \u003cli\u003ePreisner, M., Neverova-Dziopak, E., \u0026amp; Kowalewski, Z. (2021). Mitigation of eutrophication caused by wastewater discharge: A simulation-based approach. \u003cem\u003eAmbio\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(2), 413\u0026ndash;424. https://doi.org/10.1007/s13280-020-01346-4\u003c/li\u003e\n \u003cli\u003ePurba, L. D. A., Othman, F. S., Yuzir, A., Mohamad, S. E., Iwamoto, K., Abdullah, N., Shimizu, K., \u0026amp; Hermana, J. (2022). Enhanced cultivation and lipid production of isolated microalgae strains using municipal wastewater. \u003cem\u003eEnvironmental Technology and Innovation\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e. https://doi.org/10.1016/j.eti.2022.102444\u003c/li\u003e\n \u003cli\u003eRaza, N., Rizwan, M., \u0026amp; Mujtaba, G. (2024). Bioremediation of real textile wastewater with a microalgal-bacterial consortium: an eco-friendly strategy. \u003cem\u003eBiomass Conversion and Biorefinery\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(6), 7359\u0026ndash;7371. https://doi.org/10.1007/s13399-022-03214-5\u003c/li\u003e\n \u003cli\u003eRekik, I., Chaabane, Z., Missaoui, A., Bouket, A. C., Luptakova, L., Elleuch, A., \u0026amp; Belbahri, L. (2017). Effects of untreated and treated wastewater at the morphological, physiological and biochemical levels on seed germination and development of sorghum (Sorghum bicolor (L.) Moench), alfalfa (Medicago sativa L.) and fescue (Festuca arundinacea Schreb.). \u003cem\u003eJournal of Hazardous Materials\u003c/em\u003e, \u003cem\u003e326\u003c/em\u003e, 165\u0026ndash;176. https://doi.org/10.1016/j.jhazmat.2016.12.033\u003c/li\u003e\n \u003cli\u003eSadvakasova, A. K., Kossalbayev, B. D., Zayadan, B. K., Kirbayeva, D. K., Alwasel, S., \u0026amp; Allakhverdiev, S. I. (2021). Potential of cyanobacteria in the conversion of wastewater to biofuels. In \u003cem\u003eWorld Journal of Microbiology and Biotechnology\u003c/em\u003e (Vol. 37, Issue 8). Springer Science and Business Media B.V. https://doi.org/10.1007/s11274-021-03107-1\u003c/li\u003e\n \u003cli\u003eSelvaraj, D., \u0026amp; Arivazhagan, M. (2024). Synergistic effects of Spirulina platensis cultivation in textile wastewater towards nutrient removal and seed germination study. \u003cem\u003eEnvironmental Pollution\u003c/em\u003e, \u003cem\u003e357\u003c/em\u003e. https://doi.org/10.1016/j.envpol.2024.124435\u003c/li\u003e\n \u003cli\u003eSinha, S. K., Gupta, A., \u0026amp; Bharalee, R. (2016). Production of biodiesel from freshwater microalgae and evaluation of fuel properties based on fatty acid methyl ester profile. \u003cem\u003eBiofuels\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(1), 105\u0026ndash;121. https://doi.org/10.1080/17597269.2015.1118781\u003c/li\u003e\n \u003cli\u003eStanier, R. Y., Kunisawa, R., Mandel, M., \u0026amp; Cohen-Bazire, G. (1971). Purification and Properties of Unicellular Blue-Green Algae (Order Chroococcales). In \u003cem\u003eBACTEROLOGICAL REVIEWS\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eSudarshan, S., Harikrishnan, S., RathiBhuvaneswari, G., Alamelu, V., Aanand, S., Rajasekar, A., \u0026amp; Govarthanan, M. (2023). Impact of textile dyes on human health and bioremediation of textile industry effluent using microorganisms: current status and future prospects. In \u003cem\u003eJournal of Applied Microbiology\u003c/em\u003e (Vol. 134, Issue 2). Oxford University Press. https://doi.org/10.1093/jambio/lxac064\u003c/li\u003e\n \u003cli\u003eTouliabah, H. E. S., El-Sheekh, M. M., Ismail, M. M., \u0026amp; El-Kassas, H. (2022). A Review of Microalgae-and Cyanobacteria-Based Biodegradation of Organic Pollutants. In \u003cem\u003eMolecules\u003c/em\u003e (Vol. 27, Issue 3). MDPI. https://doi.org/10.3390/molecules27031141\u003c/li\u003e\n \u003cli\u003eTsolcha, O. N., Tekerlekopoulou, A. G., Akratos, C. S., Aggelis, G., Genitsaris, S., Moustaka-Gouni, M., \u0026amp; Vayenas, D. V. (2017). Biotreatment of raisin and winery wastewaters and simultaneous biodiesel production using a Leptolyngbya-based microbial consortium. \u003cem\u003eJournal of Cleaner Production\u003c/em\u003e, \u003cem\u003e148\u003c/em\u003e, 185\u0026ndash;193. https://doi.org/10.1016/j.jclepro.2017.02.026\u003c/li\u003e\n \u003cli\u003eZnad, H., Al Ketife, A. M. D., Judd, S., AlMomani, F., \u0026amp; Vuthaluru, H. B. (2018). Bioremediation and nutrient removal from wastewater by Chlorella vulgaris. \u003cem\u003eEcological Engineering\u003c/em\u003e, \u003cem\u003e110\u003c/em\u003e, 1\u0026ndash;7. https://doi.org/10.1016/j.ecoleng.2017.10.008\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"cyanobacteria, textile wastewater, bioremediation, phytotoxicity, total lipid content, biodiesel","lastPublishedDoi":"10.21203/rs.3.rs-6947833/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6947833/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study investigated the potential of two cyanobacterial species, namely \u003cem\u003eNostoc ellipsosporum\u003c/em\u003e (NE) and \u003cem\u003eSpirulina subsalsa\u003c/em\u003e (SS) to remediate textile wastewater (TWW) while obtaining wastewater-grown biomass for biodiesel production. This study reports their multi-faceted benefits using unsterilized and undiluted (100%) TWW for the first time. Both cyanobacterial species were cultivated in TWW under greenhouse conditions, focusing on their growth, TWW decolorization, pollutant removal, phytotoxicity, lipid content, and fatty acid profile. Results demonstrated significant growth and decolorization of TWW by both species, highlighting their potential for sustainable TWW treatment. NE-treated TWW (NE-TWW) achieved a chemical oxygen demand (COD) removal efficiency of 96.48%, while SS-treated TWW (SS-TWW) reached 93.11%. Ammonia removal rates were recorded at 76.28% for NE-TWW and 96.86% for SS-TWW. NE-TWW and SS-TWW achieved nitrate removal of 69% and 73%, respectively. Phosphate removal was 81.14% for NE-TWW and 33.33% for SS-TWW. Seed germination studies showed enhanced shoot and root development of green gram \u003cem\u003e(Vigna radiata)\u003c/em\u003e when irrigated with NE-TWW and SS-TWW, suggesting potential applications for irrigation. Lipid yields of 21.5% and 25% were recorded from TWW-grown biomass of NE and SS, respectively. Both species exhibited favorable fatty acid methyl ester (biodiesel) profiles, dominated by palmitic acid (C16:0), oleic acid (C18:1), stearic acid (C18:0), lauric acid (C12:0), and myristic acid (C14:0), indicating their suitability as biodiesel feedstocks. This integrated approach not only provides an effective solution for TWW treatment but also offers a sustainable feedstock for biodiesel production and an alternative water source for irrigation, aligning with the circular bioeconomy principles.\u003c/p\u003e","manuscriptTitle":"Phycoremediation potential of Nostoc ellipsosporum and Spirulina subsalsa for pollutant removal from real textile wastewater (TWW) and synchronized biodiesel production from TWW-tolerant biomass","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 11:57:59","doi":"10.21203/rs.3.rs-6947833/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":"72cf2516-2679-4ec4-9cbf-df61bca74d29","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-16T11:57:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-16 11:57:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6947833","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6947833","identity":"rs-6947833","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}