Browsing by Author "Barros, Ana"
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- 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.
- 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.
- 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.
- 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.
- Oxyfluorfen: a novel metabolic inhibitor to select microalgal chlorophyll-deficient mutant strains for nutritional applicationsPublication . Trovão dos Santos, Mafalda; Cardoso, Lucas; Schüler, Lisa; Machado, Adriana; Santo, Gonçalo Espírito; Pedroso, Humberto; Reis, Ana; Barros, Ana; Correia, Nádia; Costa, Monya; Ferreira, Sara; Cardoso, Helena; Mateus, Marília; Silva, Joana; Pereira, Hugo; Freitas, Filomena; Varela, JoãoNowadays, there is an increasing demand for novel feedstocks and alternative protein sources to meet global needs. Because of their rich nutritional profiles and high protein contents, microalgae-based food products and supplements are being developed. Nonetheless, these products present organoleptic characteristics such as taste, smell and colour that are often considered unpleasant by human and animal consumers. To address this constraint, strain improvement approaches such as random mutagenesis have been used, which combined with the right selection strategy, lead to more appealing microalgal biomass. In this work, a novel selection strategy using oxyfluorfen, an inhibitor of the chlorophyll synthesis pathway, was applied for the first time to isolate chlorophyll-deficient strains of Scenedesmus rubescens and Chlorella vulgaris upon treatment with ethyl methanesulfonate (EMS). With this approach, one S. rubescens brownish (37Y01) mutant strain, as well as two C. vulgaris mutant strains, one yellow (31Y15) and one white (31W62), were obtained. S. rubescens 37Y01 displayed a reduced protein content of 19.1% dry weight (DW) compared to that of the wildtype, which presented a protein content of 25.0% DW. C. vulgaris wildtype and mutants exhibited higher protein contents, in the 42.844.3% DW range, compared to Scenedesmus rubescens (p < 0.05). The selective pressure of this inhibitor allowed the selection of S. rubescens and C. vulgaris mutants displaying 55% and 95% decrease in chlorophyll content, respectively. The reduced chlorophyll content greatly improves the sensory properties and consumer acceptance of established mutants, increasing the potential of both strains as feedstocks to develop novel food products.
- Random mutagenesis as a promising tool for microalgal strain improvement towards industrial productionPublication . Trovão Dos Santos, Mafalda; Schüler, Lisa M.; Machado, Adriana; Bombo, Gabriel; Navalho, Sofia; Barros, Ana; Pereira, Hugo; Silva, Joana; Freitas, Filomena; Varela, JoãoMicroalgae have become a promising novel and sustainable feedstock for meeting the rising demand for food and feed. However, microalgae-based products are currently hindered by high production costs. One major reason for this is that commonly cultivated wildtype strains do not possess the robustness and productivity required for successful industrial production. Several strain improvement technologies have been developed towards creating more stress tolerant and productive strains. While classical methods of forward genetics have been extensively used to determine gene function of randomly generated mutants, reverse genetics has been explored to generate specific mutations and target phenotypes. Site-directed mutagenesis can be accomplished by employing different gene editing tools, which enable the generation of tailor-made genotypes. Nevertheless, strategies promoting the selection of randomly generated mutants avoid the introduction of foreign genetic material. In this paper, we review different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis. Current challenges hampering strain improvement, selection, and commercialization will be discussed. The combination of these approaches with high-throughput technologies, such as fluorescence-activated cell sorting, as tools to select the most promising mutants, will also be discussed.
- Scale-up and large-scale production of Tetraselmis sp CTP4 (Chlorophyta) for CO2 mitigation: from an agar plate to 100-m(3) industrial photobioreactorsPublication . Pereira, Hugo; Paramo, Jaime; Silva, Joana; Marques, Ana; Barros, Ana; Mauricio, Dinis; Santos, Tamara; Schulze, Peter; Raul J Barros; Gouveia, Luisa; Barreira, Luísa; Varela, J.Industrial production of novel microalgal isolates is key to improving the current portfolio of available strains that are able to grow in large-scale production systems for different biotechnological applications, including carbon mitigation. In this context, Tetraselmis sp. CTP4 was successfully scaled up from an agar plate to 35-and 100-m(3) industrial scale tubular photobioreactors (PBR). Growth was performed semi-continuously for 60 days in the autumn-winter season (17th October -14th December). Optimisation of tubular PBR operations showed that improved productivities were obtained at a culture velocity of 0.65-1.35 m s(-1) and a pH set-point for CO2 injection of 8.0. Highest volumetric (0.08 +/- 0.01 g L-1 d(-1)) and areal (20.3 +/- 3.2 g m(-2) d(-1)) biomass productivities were attained in the 100-m(3) PBR compared to those of the 35-m(3) PBR (0.05 +/- 0.02 g L-1 d(-1) and 13.5 +/- 4.3 g m(-2) d(-1), respectively). Lipid contents were similar in both PBRs (9-10% of ash free dry weight). CO2 sequestration was followed in the 100-m(3) PBR, revealing a mean CO2 mitigation efficiency of 65% and a biomass to carbon ratio of 1.80. Tetraselmis sp. CTP4 is thus a robust candidate for industrial-scale production with promising biomass productivities and photosynthetic efficiencies up to 3.5% of total solar irradiance.