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- Green approach for the valorization of microalgae Tetradesmus obliquusPublication . Gouveia, Luisa; Molnar Jazić, Jelena; Ferreira, Alice; Maletić, Snežana; Cvetković, Dragoljub; Vidović, Senka; Vladić, JelenaThe 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.
- Supercritical CO2 extract from Microalga Tetradesmus obliquus: the effect of high-pressure pre-treatmentPublication . Vladić, Jelena; Jerković, Igor; Radman, Sanja; Molnar Jazić, Jelena; Ferreira, Alice; Maletić, Snežana; Gouveia, LuisaHigh-pressure pre-treatment followed by supercritical carbon dioxide (ScCO2 ) extraction (300 bar, 40 ◦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 ScCO2 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.
- Modern advancement in biotechnological applications for wastewater treatment through microalgae: a reviewPublication . Goyal, Shubham; Dhanker, Raunak; Hussain, Touseef; Ferreira, Alice; Gouveia, Luisa; Kumar, Krishna; Mohamed, Heba I.Microalgae are microscopic organisms that have a broad range of applications, from wastewater treatment, CO2 mitigation to therapeutic proteins, and pharmaceuticals. Recently, the combination of wastewater treatment-based microalgae and the use of the obtained biomass as biofertilizers/stimulants/pesticides have been highly emphasized for their use in the agriculture field. Biofertilizers are a need of today's agriculture practices due to the increasing demand for food to feed a hungry planet while avoiding chemical contamination by the over-application of synthetic fertilizers. There is a constant need for modern techniques for the use of microalgae in a sustainable manner to harness their products to their full extent. Various types of bioreactors are available on the market, each with its own advantages and disadvantages, which, based on their efficiency, can be used for microalgae cultivation. This review aims at reporting recent developments in microalgae biotechnology, especially related to CO2 mitigation, wastewater purification, biofuel, feedstock, future food, therapeutic proteins, pharmaceuticals, and biofertilizers, highlighting some of the current research in this field and future development priorities.
- From piggery wastewater to wheat using microalgae towards zero wastePublication . Ferreira, Alice; Figueiredo, Daniel; Ferreira, Francisca; Marujo, Ana; Bastos, Carolina; Martin-Atanes, Guillermo; Ribeiro, Belina; Štěrbová, Karolína; Marques-dos-Santos, Cláudia; Acién, F. Gabriel; Gouveia, LuisaMicroalgae production is still expensive, driving the need to lower costs while strengthening the industry's environmental sustainability. Microalgae are recognized tools for efficient wastewater treatment, offering the recycling of nutrients and water for agriculture, and producing biomass rich in growth-promoting compounds to improve plant productivity and resistance to adverse conditions. The use of wastewater can reduce cultivation costs as it is a source of nutrients and water. Alternative low-cost methods can significantly decrease harvesting costs, which represents one of the most expensive steps of the whole process.The goal of this work was to evaluate the potential of wastewater-grown microalga biomass for agriculture purposes. To reduce production costs, the microalga Tetradesmus obliquus was produced in pre-treated photo-Fenton (PF) piggery wastewater in combination with the use of different harvesting techniques - electro-coagulation, flocculation, and centrifugation, and different combinations. From the wastewater treatment pro-cess, two fractions (biomass and supernatant) were evaluated for germination and growth of wheat (Triticum aestivum L.) plants and compared to non-harvested microalga culture (MC), distilled water, and Hoagland (synthetic) solution. The concentrated resulting from PF was also tested as a biofertilizer.The results confirm that both biomass and supernatants are useful for agricultural applications. The obtained biomass elicited a 20-105 % increase in germination index compared to the control, while supernatants were inhibiting. The opposite trend was observed at later stages of wheat growth, where the nutrient-enriched su-pernatants and the PF concentrate (PF-CC) increased the number of tillers (3-5) and leaves (30-42) after 83 days. Wheat plants treated with MC and PF-CC produced similar number of ears (3.4 & PLUSMN; 0.5 and 6.0 & PLUSMN; 4.1 ears per plant, respectively) than the synthetic control (5.7 & PLUSMN; 1.4) after 182 days. All fractions obtained from the process can be used in a zero-waste process.
- Application of green technology to extract clean and safe bioactive compounds from tetradesmus obliquus biomass grown in poultry wastewaterPublication . Vladić, Jelena; Jazić, Jelena Molnar; Ferreira, Alice; Maletić, Snežana; Cvetković, Dragoljub; Agbaba, Jasmina; Vidović, Senka; Gouveia, LuisaMicroalgae 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.
- Rhodosporidium toruloides and Tetradesmus obliquus populations dynamics in symbiotic cultures, developed in brewery wastewater, for lipid productionPublication . Dias, Carla; Gouveia, Luisa; Santos, José A. L.; Reis, Alberto; da Silva, Teresa LopesIn this work, primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) separately, or mixed at the ratios of 1:1 (PBWW:SBWW) and 1:7 (PBWW:SBWW), with or without supplementation with sugarcane molasses (SCM), were used as culture media for lipid production by a mixed culture of the oleaginous yeast Rhodosporidium toruloides NCYC 921 and the microalgae Tetradesmus obliquus (ACOI 204/07). Flow cytometry was used to understand the dynamics of the two micro-organisms during the mixed cultures evolution, as well as to evaluate the physiological states of each microorganism, in order to assess the impact of the different brewery effluent media composition on the microbial consortium performance. Both brewery wastewaters (primary and secondary) without supplementation did not allow R. toruloides heterotrophic growth. Nevertheless, all brewery wastewater media, with and without SCM supplementation, allowed the microalgae growth, although the yeast was the dominant population. The maximum total biomass concentration of 2.17 g L-1 was achieved in the PBWW mixed cultivation with 10 g L-1 of SCM. The maximum lipid content (14.86% (w/w DCW)) was obtained for the mixed culture developed on SBWW supplemented with 10 g L-1 of SCM. This work demonstrated the potential of using brewery wastewater supplemented with SCM as a low-cost culture medium to grow R. toruloides and T. obliquus in a mixed culture for brewery wastewater treatment with concomitant lipid production.
- Exploring different pretreatment methodologies for allowing microalgae growth in undiluted piggery wastewaterPublication . Ferreira, Alice; Figueiredo, Daniel; Cardeiras, Rodrigo; Nabais, Rui; Ferreira, Francisca; Ribeiro, Belina; Cordovil, Cláudia M. d. S.; Acién, F. Gabriel; Gouveia, LuisaThe overapplication of manure on agricultural soils leads to nitrogen and phosphorus discharge into the aquatic environment, resulting in serious eutrophication problems and decreased water quality. Piggery wastewater (PWW) can be treated by microalgae to recycle nutrients, but the toxic levels of ammonia and organic matter hinder their growth. Fresh water is usually used to dilute PWW, but it is a scarce resource. The implementation of a pretreatment step before microalgae-based treatment could make PWW suitable for microalgae growth. Electrocoagulation, ammonia stripping, photo-Fenton, and constructed wetlands were evaluated as pretreatment methods to reduce ammonia, chemical oxygen demand (COD), color, and total suspended solids. Moreover, the pretreated PWWs were tested to grow the microalga Tetradesmus obliquus. Photo-Fenton showed the best results among the other pretreatments, achieving removal efficiencies above 90%, except for ammonia. This resulted in T. obliquus being capable of growing on undiluted PWW, even at higher ammonia levels, achieving similar biomass productivity to synthetic medium (66.4 ± 17.8 mg·L−1·day−1 and 60.1 ± 10.4 mg·L−1·day−1, respectively) almost doubling with pH control (116.5 mg·L−1·day−1). Thus, this pretreatment seems to be the most promising one to incorporate into microalgae-based treatment systems and must be further explored.
- Valorisation of microalga Tetradesmus obliquus grown in brewery wastewater using subcritical water extraction towards zero wastePublication . Ferreira, Alice; Molnar Jazić, Jelena; Gouveia, Luisa; Maletić, Snežana; Tomić, Milan; Agbaba, Jasmina; Vladić, Jelenan 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.
- A circular approach for landfill leachate treatment: Chemical precipitation with biomass ash followed by bioremediation through microalgaePublication . Viegas, Catarina; Nobre, Catarina; Mota, Andre; Vilarinho, Candida; Gouveia, Luisa; Goncalves, MargaridaThe aim of this work was to study an integrated approach for landfill leachate remediation comprising chemical precipitation with biomass bottom ash as a pre-treatment to reduce color and turbidity followed by bioremediation through microalgae treatment for effluent disposal. Optimal pre-treatment conditions were determined through batch experiments and were found to be 160 g L-1 ash dose, 96 h of contact time, overhead agitation at 15 rpm and ash particle size below 500 mu m. These conditions led to removal efficiencies of 74.3% for chemical oxygen demand and 98.5% for color. Large quantities of sludge containing excess biomass ash and precipitated compounds were formed during the pre-treatment. To minimize solid disposal, this sludge was tested as a raw material for cementitious and aggregate substitute in mortar formulations. Following the pre-treatment, the leachate was inoculated with six different microalgae species to evaluate their ability to grow in such a recalcitrant effluent and remediate it. After a period of 27 days biomass concentration from 0.4 to 1.2 g L-1 were achieved for the tested microalgae. Removal efficiencies were in the range of 18-62% for COD, 63-71% for N, and 15-100% for P. At the end of the treatment, algal biomass was characterized regarding protein, lipid, fatty acids, carbohydrate, and ash contents. This approach allows a low-cost remediation of these recalcitrant effluents when compared with the present options that include inverse osmosis, and the valorization of ash-rich precipitates and microalgae biomass improves the sustainability of the overall process.
- Heterotrophic and photoautotrophic media optimization using response surface methodology for the Novel Microalga Chlorococcum amblystomatisPublication . Correia, Nádia; Pereira, Hugo; Schulze, Peter S.C.; Costa, Monya; Santo, Gonçalo E.; Guerra, Inês; Trovão dos Santos, Mafalda; Barros, Ana; Cardoso, Helena; Silva, Joana L.; Gouveia, Luisa; Varela, JoãoThe nutritional requirements of novel microalgal strains are key for their effective cultivation and metabolite content. Therefore, the optimization of heterotrophic and photoautotrophic culture media is crucial for novel Chlorococcum amblystomatis growth. Heterotrophic and photoautotrophic biomass samples were characterized to identify the differences between their heterotrophic and photoautotrophic biomass composition and their biotechnological potential. Media optimization through surface response methodology led to 44.9 and 51.2% increments in C. amblystomatis-specific growth rates under heterotrophic and photoautotrophic growth, respectively. This microalga registered high protein content (61.49–73.45% dry weight), with the highest value being observed in the optimized photoautotrophic growth medium. The lipid fraction mainly constituted polyunsaturated fatty acids, ranging from 44.47 to 51.41% for total fatty acids (TFA) in cells under heterotrophy. However, these contents became significantly higher (70.46–72.82% TFA) in cultures cultivated under photoautotrophy. An interesting carotenoids content was achieved in the cultures grown in optimized photoautotrophic medium: 5.84 mg·g−1 β-carotene, 5.27 mg·g−1 lutein, 3.66 mg·g−1 neoxanthin, and 0.75 mg·g−1 violaxanthin. Therefore, C. amblystomatis demonstrated an interesting growth performance and nutritional profile for food supplements and feed products that might contribute to meeting the world’s nutritional demand.
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