Browsing by Author "Silva, Joana"
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- Bioprospecting for industrially relevant exopolysaccharide-producing cyanobacteria under portuguese simulated climatePublication . Cruz, José Diogo; Delattre, Cédric; Felpeto, Aldo Barreiro; Pereira, Hugo; Pierre, Guillaume; Morais, João; Petit, Emmanuel; Silva, Joana; Azevedo, Joana; Elboutachfaiti, Redouan; Maia, Inês Beatriz; Dubessay, Pascal; Michaud, Philippe; Vasconcelos, VitorCyanobacterial exopolysaccharides (EPS) are potential candidates for the production of sustainable biopolymers. Although the bioactive and physicochemical properties of cyanobacterial-based EPS are attractive, their commercial exploitation is limited by the high production costs. Bioprospecting and characterizing novel EPS-producing strains for industrially relevant conditions is key to facilitate their implementation in various biotechnological applications and fields. In the present work, we selected twenty-five Portuguese cyanobacterial strains from a diverse taxonomic range (including some genera studied for the first time) to be grown in diel light and temperature, simulating the Portuguese climate conditions, and evaluated their growth performance and proximal composition of macronutrients. Synechocystis and Cyanobium genera, from marine and freshwater origin, were highlighted as fast-growing (0.1-0.2 g L-1 day(-1)) with distinct biomass composition. Synechocystis sp. LEGE 07367 and Chroococcales cyanobacterium LEGE 19970, showed a production of 0.3 and 0.4 g L-1 of released polysaccharides (RPS). These were found to be glucan-based polymers with high molecular weight and a low number of monosaccharides than usually reported for cyanobacterial EPS. In addition, the absence of known cyanotoxins in these two RPS producers was also confirmed. This work provides the initial steps for the development of cyanobacterial EPS bioprocesses under the Portuguese climate.
- Chrysotila pseudoroscoffensis as a source of high-value polar lipids with antioxidant activity: A lipidomic approachPublication . Moreira, Ana S. P.; Gonçalves, Joana; Conde, Tiago A.; Couto, Daniela; Melo, Tânia; Maia, Inês Beatriz; Pereira, Hugo; Silva, Joana; Domingues, M. Rosário; Nunes, CláudiaMicroalgae are emerging as sustainable sources of a wide range of high-value compounds. However, the knowledge about microalgae polar lipids is still limited, despite their interest due to their chemical diversity and bioactivity. This study shows, for the first time, the polar lipidome of the haptophyte microalga Chrysotila pseudoroscoffensis unveiled by using hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS and MS/MS) and gas chromatography-mass spectrometry (GC-MS). Freeze-dried C. pseudoroscoffensis biomass has a lipid content of 6.4 %, containing higher amounts of ash (45.5 %), proteins (11.6 %), and sugars (11.0 %). Uronic acids (53.8 mol%) are the sugars present in higher content, followed by glucose (13.7 mol%) and galactose (12.7 mol%). The polar lipid species identified by HILIC-MS/MS included betaine lipids, glycolipids, and phospholipids, some of them with recognised bioactive properties. Among the lipid classes found from C. pseudoroscoffensis, some are less reported in algae: betaine lipids diacylglycerylcarboxyhydroxymethylcholine (DGCC) and monoacylglycerylcarboxyhydroxymethylcholine (MGCC) (characteristic of haptophyte microalgae); acid glycolipid class glucuronosyldiacylglycerol (GlcADG) (mainly reported in plants with protective effects in phosphate-deprivation conditions); and phospholipid classes monomethylphosphatidylethanolamine (MMPE) and lysomonomethylphosphatidylethanolamine (MMLPE). As estimated by colorimetric assays, glycolipids and phospholipids accounted for 64 and 3 % of the total lipid extracts, respectively. Fatty acid profiling by GC-MS showed that total esterified fatty acids accounted for about 32 % of the total lipid extracts, of which 23 % were omega-3 polyunsaturated fatty acids (PUFA). Four lipid extract concentrations (12.5, 62.5, 125 and 250 mu g mL-1 in ethanol) were tested and displayed antioxidant capacity toward 2,2 & PRIME;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. A dose-dependent behaviour was observed with IC50 of 111.9 mu g mL(-1) for ABTS and IC35 of 234.8 mu g mL(-1) for DPPH assay. In conclusion, the lipid extracts of C. pseudoroscoffensis may be a source of high-value lipids for the development of novel microalgae-based products, namely nutraceuticals and cosmeceuticals.
- Development of an organic culture medium for autotrophic production of chlorella vulgaris biomassPublication . Machado, Adriana; Pereira, Hugo; Costa, Margarida; Santos, Tamara; Carvalho, Bernardo; Soares, Maria; Quelhas, Pedro; Silva, Joana T.; Trovão, Mafalda; Barros, Ana; Varela, João; Vicente, António A.; Silva, JoanaMicroalgal biomass has gained increasing attention in the last decade for various biotechnological applications, including human nutrition. Certified organic products are currently a growing niche market in which the food industry has shown great interest. In this context, this work aimed at developing a certified organic culture medium for the production of autotrophic Chlorella vulgaris biomass. A preliminary assay in 2 L bubble column photobioreactors was performed in order to screen di erent commercial organic substrates (OS) at a normalized concentration of N (2 mmol L1). The highest growth performance was obtained using EcoMix4 and Bioscape which showed similar biomass concentrations compared to the synthetic culture medium (control). In order to meet the nutrient needs of Chlorella, both OS underwent elemental analyses to assess their nutrient composition. The laboratory findings allowed the development of a final organic culture medium using a proportion of Bioscape/EcoMix4 (1:1.2, m/m). This organic culture medium was later validated outdoors in 125 L flat panel and 10 m3 tubular flow through photobioreactors. The results obtained revealed that the developed organic medium led to similar microalgal growth performance and biochemical composition of produced biomass, as compared to the traditional synthetic medium. Overall, the formulated organic medium was e ective for the autotrophic production of organic C. vulgaris biomass.
- Effects of outdoor and indoor cultivation on the polar lipid composition and antioxidant activity of Nannochloropsis oceanica and Nannochloropsis limnetica: A lipidomics perspectivePublication . Couto, Daniela; Conde, Tiago A.; Melo, Tânia; Neves, Bruna; Costa, Margarida; Cunha, Pedro; Guerra, Inês; Correia, Nádia; Silva, Joana T.; Pereira, Hugo; Varela, João; Silva, Joana; Domingues, Rosário; Domingues, PedroNannochloropsis is a genus of eicosapentaenoic acid-rich microalgae with high levels of value-added polar lipids. However, the polar lipid composition of microalgal biomass is highly dependent on culture conditions (e.g., light or temperature), which are significantly different under indoor and outdoor culture conditions. In this study, we sought to investigate the plasticity of the polar lipid profile of a marine (N. oceanica) and a freshwater (N. limnetica) species of Nannochloropsis grown in indoor and outdoor photobioreactors. To this end, the polar lipidome and fatty acid profiles were characterized by liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS), respectively. In addition, the antioxidant activity of their lipid extracts was assessed. The highest lipid contents were obtained for the two species grown indoors. LC-MS analysis identified 239 different polar lipid species, of which 220 were shared by all experimental groups. Candidate lipid biomarkers from both culture systems were proposed, including MGDG(34:2), MGDG(34:1) and PG(36:6). For both species, indoor conditions lead to lipid extracts rich in glycolipids and higher in oleic acid content. In contrast, outdoor conditions lead to higher proportions of phospholipids and betaine lipids and a higher relative content of eicosapentaenoic acid (EPA). The polar lipid profile of the two Nannochloropsis species differed primarily in the relative amounts of certain betaine lipids, mainly DGTS (which was increased in N. oceanica) and lysolipids (LPC, and LPE) (increased in N. limnetica), although the majority of lipids were observed in both species. The lipid extracts showed antioxidant activity (IC15) ranging from 30.4 +/- 1.8 to 45.7 +/- 1.6 mu mol Trolox g-1 of lipid extract. Overall, this study provides insight into the lipid metabolic adaptation of two Nannochloropsis species, providing the know-how to obtain a healthy polar lipid-rich biomass useful for novel applications in pharmaceutical, nutraceutical, or novel foods.
- Growth performance, biochemical composition and sedimentation velocity of Tetraselmis sp. CTP4 under different salinities using low-cost lab- and pilot-scale systemsPublication . Trovão, Mafalda; Pereira, Hugo; Silva, Joana; Páramo, Jaime; Quelhas, Pedro; Santos, Tamara; Silva, Joana T.; Machado, Adriana; Gouveia, Luísa; Barreira, Luísa; Varela, JoãoBiomass harvesting is one of the most expensive steps of the whole microalgal production pipeline. Therefore, the present work aimed to understand the effect of salinity on the growth performance, biochemical composition and sedimentation velocity of Tetraselmis sp. CTP4, in order to establish an effective low-cost pilot-scale harvesting system for this strain. At lab scale, similar growth performance was obtained in cultures grown at salinities of 5, 10 and 20 g L-1 NaCl. In addition, identical settling velocities (2.4-3.6 cm h-1) were observed on all salinities under study, regardless of the growth stage. However, higher salinities (20 g L-1) promoted a significant increase in lipid contents in this strain compared to when this microalga was cultivated at 5 or 10 g L-1 NaCl. At pilot-scale, cultures were cultivated semi-continuously in 2.5-m3 tubular photobioreactors, fed every four days, and stored in a 1-m3 harvesting tank. Upon a 24-hour settling step, natural sedimentation of the microalgal cells resulted in the removal of 93% of the culture medium in the form of a clear liquid containing only vestigial amounts of biomass (0.07 ± 0.02 g L-1 dry weight; DW). The remaining culture was recovered as a highly concentrated culture (19.53 ± 4.83 g L-1 DW) and wet microalgal paste (272.7 ± 18.5 g L-1 DW). Overall, this method provided an effective recovery of 97% of the total biomass, decreasing significantly the harvesting costs.
- Heterotrophic cultivation of Chlorella vulgaris yellow mutant on sidestreams: Medium formulation and process scale-upPublication . Trovão, Mafalda; Barros, Ana; Machado, Adriana; Reis, Ana; Pedroso, Humberto; Espírito Santo, Gonçalo; Correia, Nádia; Costa, Monya; Ferreira, Sara; Varela, João; Cardoso, Helena; Silva, Joana; Pereira, Hugo; Freitas, FilomenaMicroalgal protein is a promising feedstock to complement and/or replace other protein sources. Besides requiring less land and water usage, microalgae production is a more sustainable process, especially if industrial sidestreams are used as nutrient sources. Additionally, the heterotrophic cultivation of microalgae, such as Chlorella vulgaris, enables the achievement of much higher biomass productivity and lower areal footprint than autotrophic cultivation. Chlorophyll-deficient strains of C. vulgaris, as the yellow strain 7Y, provide microalgal biomass with improved sensory properties. In line with this, a waste-based medium was formulated to cultivate this strain, aiming at maximum biomass productivity. In this context, several industrial sidestreams were screened, and two food wastes and corn molasses were selected for their high nitrogen and glucose concentrations, respectively. The waste-based medium formulated was compared to the inorganic optimised medium at laboratory scale in Erlenmeyer flasks and 7-L reactors. The results obtained in the 7-L fermenters revealed that both conditions achieved similar biomass productivities and growth rates of approximately 14 g L-1 d-1 and 0.8 d-1, respectively. The biomass and protein productivities were further enhanced by supplying a higher nitrogen concentration in the feeding solution when the process was scaled-up to 200-L reactors, reaching 22 and 6.3 g L-1 d-1, respectively, thus validating the developed industrial waste-based medium for the efficient cultivation of C. vulgaris under heterotrophic conditions.
- Innovation for resiliencePublication . Nunes, Adélia N.; Serrano, António; Sequeira, Bernardete; Marques, João Filipe; Jesus, Bruno; Alves, Carolina; Ferreira, Célia Marisa Fonseca; Pinto, Hugo; Silva, Joana; Teixeira, José Afonso; Barata, José Monteiro; Grave, Luís S.; Pereira, Margarida; Cesário, Marisa; Guerra, Paula; Tomé, Ricardo; Fernandes, Sílvia; Marques, Teresa Sá; Noronha, Teresa de; Álvarez, Victória del Pino; Noronha, Teresa de; Pinto, HugoLast decade is characterized by different types of crises and shocs in the socioeconomic systems, creating a turbulent context and calling for a better understanding of what the dynamic perspective of change is. For countries, regions and cities a better understanding of governance urges and calls for action.
- Isolation and characterization of novel chlorella vulgaris mutants with low chlorophyll and improved protein contents for food applicationsPublication . Lisa Schueler, Lisa M. Schueler; Morais, Etiele Greque de; Trovão, Mafalda; Machado, Adriana; Carvalho, Bernardo; Carneiro, Mariana; Maia, Inês; Soares, Maria; Duarte, Paulo; Barros, Ana; Pereira, Hugo; Silva, Joana; Varela, JoãoMicroalgae are widely used as food supplements due to their high protein content, essential fatty acids and amino acids as well as carotenoids. The addition of microalgal biomass to food products (e.g., baked confectioneries) is a common strategy to attract novel consumers. However, organoleptic factors such as color, taste and smell can be decisive for the acceptability of foods supplemented with microalgae. The aim of this work was to develop chlorophyll-deficient mutants of Chlorella vulgaris by chemically induced random mutagenesis to obtain biomass with different pigmentations for nutritional applications. Using this strategy, two C. vulgaris mutants with yellow (MT01) and white (MT02) color were successfully isolated, scaled up and characterized. The changes in color of MT01 and MT02 mutant strains were due to an 80 and 99% decrease in their chlorophyll contents, respectively, as compared to the original wild type (WT) strain. Under heterotrophic growth, MT01 showed a growth performance similar to that of the WT, reaching a concentration of 5.84 and 6.06 g L-1, respectively, whereas MT02 displayed slightly lower growth (4.59 g L-1). When grown under a light intensity of 100 μmol m-2 s-1, the pigment content in MT01 increased without compromising growth, while MT02 was not able to grow under this light intensity, a strong indication that it became light-sensitive. The yellow color of MT01 in the dark was mainly due to the presence of the xanthophyll lutein. On the other hand, phytoene was the only carotenoid detected in MT02, which is known to be colorless. Concomitantly, MT02 contained the highest protein content, reaching 48.7% of DW, a 60% increase as compared to the WT. MT01 exhibited a 30% increase when compared to that of the WT, reaching a protein content of 39.5% of DW. Taken together, the results strongly suggest that the partial abrogation of pigment biosynthesis is a factor that might promote higher protein contents in this species. Moreover, because of their higher protein and lower chlorophyll contents, the MT01 and MT02 strains are likely candidates to be feedstocks for the development of novel, innovative food supplements and foods.
- Isolation and selection of protein-rich mutants of chlorella vulgaris by fluorescence-activated cell sorting with enhanced biostimulant activity to germinate garden cress seedsPublication . Trovão dos Santos, Mafalda; Schüler, Lisa; Pedroso, Humberto; Reis, Ana; Santo, Gonçalo Espírito; Barros, Ana; Correia, Nádia; Ribeiro, Joana; Bombo, Gabriel; Gama, Florinda; Viana, Catarina; Costa, Monya; Ferreira, Sara; Cardoso, Helena; Varela, João; Silva, Joana; Freitas, Filomena; Pereira, HugoMicroalgae are a promising feedstock with proven biostimulant activity that is enhanced by their biochemical components (e.g., amino acids and phytohormones), which turns them into an appealing feedstock to reduce the use of fertilisers in agriculture and improve crop productivity and resilience. Thus, this work aimed to isolate protein-rich microalgal mutants with increased biostimulant activity. Random mutagenesis was performed with Chlorella vulgaris, and a selection of protein-rich mutants were sorted through fluorescence-activated cell sorting (FACS), resulting in the isolation of 17 protein-rich mutant strains with protein contents 19-34% higher than that of the wildtype (WT). Furthermore, mutant F4 displayed a 38%, 22% and 62% higher biomass productivity, growth rate and chlorophyll content, respectively. This mutant was then scaled up to a 7 L benchtop reactor to produce biomass and evaluate the biostimulant potential of this novel strain towards garden cress seeds. Compared to water (control), the germination index and the relative total growth increased by 7% and 19%, respectively, after the application of 0.1 g L-1 of this bioproduct, which highlights its biostimulant potential.
- Isolation, identification and biotechnological applications of a novel, robust, free-living Chlorococcum (Oophila) amblystomatis strain isolated from a local pondPublication . Correia, Nádia; Pereira, Hugo; Silva, Joana T.; Santos, Tamara; Soares, Maria; Sousa, Carolina B.; Schüler, Lisa Maylin; Costa, Margarida; J. C. or Varela J. or Varela J.C.S.; Pereira, Leonel; Silva, JoanaBioprospection of novel autochthonous strains is key to the successful industrial-scale production of microalgal biomass. A novel Chlorococcum strain was recently isolated from a pond inside the industrial production facility of Allmicroalgae (Leiria, Portugal). Phylogenetic analysis based on 18S ribosomal ribonucleic acid (rRNA) gene sequences suggests that this isolate is a novel, free-living Oophila amblystomatis strain. However, as our phylogenetic data strongly suggests that the aforementioned taxon belongs to the genus Chlorococcum, it is here proposed to rename this species as Chlorococcum amblystomatis. In order to characterize the biotechnological potential of this novel isolate, growth performance and biochemical composition were evaluated from the pilot (2.5-m3) to industrial (10-m3) scale. The highest maximum areal productivity (36.56 g m2 day1) was reached in a 10-m3 tubular photobioreactor (PBR), as compared to that obtained in a 2.5-m3 PBR (26.75 g m2 day1). Chlorococcum amblystomatis displayed high protein content (48%–56% dry weight (DW)) and moderate levels of total lipids (18%–31% DW), carbohydrates (6%–18% DW) and ashes (9%–16% DW). Furthermore, the lipid profile was dominated by polyunsaturated fatty acids (PUFAs). The highest pigment contents were obtained in the 2.5-m3 PBR, where total chlorophylls accounted for 40.24 mg g1 DW, followed by lutein with 5.37 mg g1 DW. Overall, this free-living Chlorococcum amblystomatis strain shows great potential for nutritional applications, coupling a promising growth performance with a high protein content as well as relevant amounts of PUFAs, chlorophyll, and carotenoids.
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