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- Green approach for the valorization of microalgae Tetradesmus obliquusPublication . Gouveia, Luisa; Jazic, Jelena Molnar; Ferreira, Alice; Maletic, Snezana; Cvetkovic, Dragoljub; Vidovic, Senka; Vladic, JelenaABSTRACT: The main goal of this study was to develop an efficient, green approach for the valorization of Tetradesmus obliquus biomass, with zero waste. This microalga was selected because it is widespread, resistant, easy for cultivation, and fast-growing. In the first step, supercritical carbon dioxide (ScCO2) extraction followed by rapid gas decompression was used for the extraction of biomass. The following step was to apply ultrasound-assisted (UA), microwave-assisted (MA), and subcritical water (SW) extraction on the ScCO2-treated biomass to determine the most efficient processing technology. SW demonstrated to be a superior technique over MW and UA with regard to extraction yield and antioxidant content. Moreover, the chemical and microbiological profiles of SW extracts were determined to evaluate their potential and safety. In addition, to create a procedure with zero waste, the solid waste after SW extraction (residue) was analyzed. The organic profile of extracts and residues contained compounds that belong to groups of aliphatic saturated hydrocarbons, aliphatic unsaturated hydrocarbons, alkylated hydrocarbons, ketones, phenols, and esters. Furthermore, these compounds can be applied in different industries including the pharmaceutical and cosmetic industries. Additionally, the content of metals in residues indicated that this material can be used as animal feed and in agriculture. Finally, a complete reduction of microorganisms present in the initial biomass was obtained for the extracts and residues, indicating their safety.
- Application of Green Technology to Extract Clean and Safe Bioactive Compounds from Tetradesmus obliquus Biomass Grown in Poultry WastewaterPublication . Vladic, Jelena; Jazic, Jelena Molnar; Ferreira, Alice; Maletic, Snezana; Cvetkovic, Dragoljub; Agbaba, Jasmina; Vidovic, Senka; Gouveia, LuisaABSTRACT: Microalgae are capable of assimilating nutrients from wastewater (WW), producing clean water and biomass rich in bioactive compounds that need to be recovered from inside the microalgal cell. This work investigated subcritical water (SW) extraction to collect high-value compounds from the microalga Tetradesmus obliquus after treating poultry WW. The treatment efficiency was evaluated in terms of total Kjeldahl nitrogen (TKN), phosphate, chemical oxygen demand (COD) and metals. T. obliquus was able to remove 77% TKN, 50% phosphate, 84% COD, and metals (48-89%) within legislation values. SW extraction was performed at 170 degrees C and 30 bar for 10 min. SW allowed the extraction of total phenols (1.073 mg GAE/mL extract) and total flavonoids (0.111 mg CAT/mL extract) with high antioxidant activity (IC50 value, 7.18 mu g/mL). The microalga was shown to be a source of organic compounds of commercial value (e.g., squalene). Finally, the SW conditions allowed the removal of pathogens and metals in the extracts and residues to values in accordance with legislation, assuring their safety for feed or agriculture applications.
- Innovative approach in sustainable agriculture: Harnessing microalgae potential via subcritical water extractionPublication . Ferreira, Alice; Vladic, Jelena; Corrêa, D. O.; Butzke, Valéria Louzada Leal; Martins, Pedro L.; Ribeiro, Belina; Santos, Cláudia Marques dos; Acién, F. Gabriel; Gouveia, LuisaABSTRACT: Microalgae can contribute to sustainable agriculture and wastewater treatment. This study investigated Tetradesmus obliquus, grown in piggery wastewater (To-PWW), as a biostimulant/biofertilizer compared to biomass grown in synthetic medium (To-B). Subcritical water extraction was tested for disruption/hydrolysis of wet biomass, at three temperatures (120, 170, and 220 °C) and two biomass loads (1:10 and 1:80 (g dry biomass/mL water)). Extracts were evaluated for germination, and root formation/expansion. Residues were quantified for nutrient composition to assess their biofertilizer potential and tested for their affinity to oil compounds for bioremediation. The best germination was achieved by To-B extracts at 170 °C (1:10: 148 % at 0.2 g/L, 1:80: 145 % at 0.5 g/L). Only To-PWW extracts at 0.2 g/L had a significant germination effect (120 °C: 120–123 % for both loads; 170 °C: 115 % for 1:80). To-PWW extract at 120 °C and 1:10 significantly affected cucumber and mung bean root formation (224 and 268 %, respectively). Most extracts significantly enhanced root expansion, with all To-B extracts at 1:10 showing the best results (139–181 %). The residues contained essential nutrients (NPK), indicating their biofertilizer potential, helping decrease synthetic fertilizers demands. To-B residues had high affinity to toluene and diesel but lower to used cooking and car oils. To-PWW showed very low affinity to all oil compounds. Finally, all residues were only able to form stable emulsions with the used car oil. This study fully exploits the use of microalgal biomass in sustainable agriculture, producing biostimulant extracts, and residues for biofertilizer and bioremediation, from a low-cost wastewater source.
- Supercritical CO2 extract from microalga Tetradesmus obliquus: the effect of high-pressure pre-treatmentPublication . Vladic, Jelena; Jerkovic, Igor; Radman, Sanja; Jazic, Jelena Molnar; Ferreira, Alice; Maletic, Snezana; Gouveia, LuisaABSTRACT: High-pressure pre-treatment followed by supercritical carbon dioxide (ScCO2) extraction (300 bar, 40 degrees C) was applied for the attainment of the lipophilic fraction of microalga Tetradesmus obliquus. The chemical profile of supercritical extracts of T. obliquus was analyzed by ultra-highperformance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). Moreover, the impact of ScCO(2 )on the microbiological and metal profile of the biomass was monitored. The application of the pre-treatment increased the extraction yield approximately three-fold compared to the control. In the obtained extracts (control and pre-treated extracts), the identified components belonged to triacylglyceroles, fatty acid derivatives, diacylglycerophosphocholines and diacylglycerophosphoserines, pigments, terpenes, and steroids. Triacylglycerols (65%) were the most dominant group of compounds in the control extract. The pre-treatment decreased the percentage of triacylglycerols to 2%, while the abundance of fatty acid derivatives was significantly increased (82%). In addition, the pre-treatment led to an increase in the percentages of carotenoids, terpenoids, and steroids. Furthermore, it was determined that ScCO2 extraction reduced the number of microorganisms in the biomass. Considering its microbiological and metal profiles, the biomass after ScCO2 can potentially be used as a safe and important source of organic compounds.
- Clean production of microalgae high-value lipid fraction: Influence of different pretreatments on chemical and cytotoxic profiles of Chlorella vulgaris supercritical extracts and life cycle assessmentPublication . Vladic, Jelena; Radman, Sanja; Jerkovic, Igor; Besu, Irina; Speranza, Lais Galileu; Hala, Ahmad Furqan; Kovacevic, Strahinja; Perreira, Hugo; Gouveia, LuisaABSTRACT: Microalgae have emerged as a promising natural resource rich in bioactive compounds. Health-beneficial properties of microalgae, coupled with advantageous characteristics such as high biomass productivity, adaptability, robustness, and carbon dioxide mitigation, position them as a viable solution for global sustainable food production. This study explored clean and environmentally friendly processes to enhance the recovery of lipid bioactive fractions. Microwave (MW), enzymatic (ENZ), and ultrasound (US) pretreatments were applied to improve environmentally friendly extraction of lipid-based components using supercritical CO2. The effects of these pretreatments on extraction yield, chemical profiles, and cytotoxic properties of Chlorella vulgaris (Cv) and smooth C. vulgaris (sCv) extracts were investigated. Additionally, a Life Cycle Assessment (LCA) was conducted to evaluate environmental impacts. MW pretreatment achieved the highest yield increases, from 2.58 times (Cv) to 3.15 times (sCv). UHPLC-ESI-HRMS analysis revealed shifts in the distribution of pigments and derivatives caused by pretreatments, with ENZ extracts showing the most pronounced changes: pigments increased from 9.24% (control Cv) to 40.92% (Cv) and from 12.52% (control sCv) to 71.12% (sCv). Cv extracts exhibited greater activity against MDA-MB-453 cells, while sCv extracts from US pretreatment demonstrated the strongest effect on HeLa cells. The LCA indicated reduced environmental impacts of the pretreatment-enhanced processes up to 65% compared to the control. A scenario analysis was presented to show further possible impact reduction by recirculating the CO2 solvent and substituting the energy source. These findings provide valuable insights into sustainable and scalable green processes for recovering microalgal bioactive components.
- Valorisation of microalga Tetradesmus obliquus grown in brewery wastewater using subcritical water extraction towards zero wastePublication . Ferreira, Alice; Jazic, Jelena Molnar; Gouveia, Luisa; Maletic, Snezana; Tomic, Milan; Agbaba, Jasmina; Vladic, JelenaABSTRACT: In this study, green technology was applied for extraction of compounds from wastewater-grown microalga biomass with the final goal of obtaining microbiologically safe products within a sustainable biorefinery process with zero waste. Tetradesmus obliquus biomass resulting from brewery wastewater treatment, with (To-CO2) and without CO2 supplementation (To), was submitted to subcritical water extraction (SWE) at temperatures 120-220 degrees C for 10 min. The impact of the different SWE conditions in the obtained liquid extracts and solid residues were investigated for metal content and chemical and microbiological profiles. Gas chromatography-mass spectrometry analysis indicated that of T. obliquus extracts and residues are valuable sources of aliphatic saturated, unsaturated, and alkylated (mostly methylated) hydrocarbons, phenols, esters, and ketones. Polyphenolic content and antioxidant activity were enhanced approximately 4 times by increasing the temperature from 120 to 220 degrees C. Also, the content of polyphenols doubled when T. obliquus was supplemented with CO2 for all the tested temperatures (To: 0.249-1.016 mg GAE mL(-1); To-CO2: 0.437 - 1.767 mg GAE mL(-1)). The microbiological analysis determined that liquid extracts and residues represent safe sources of bioactive components that can be used in different industries. In addition, the lower content of heavy metals in residues suggests the possibility of using the solid waste as animal feed or soil conditioner in agricultural applications.