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- Two-stage lipid induction in the microalga tetraselmis striata CTP4 upon exposure to different abiotic stressesPublication . Monteiro, Ivo; Schüler, Lisa M.; Santos, Eunice; Pereira, Hugo; Schulze, Peter S.C.; Florindo, Claudia; Varela, João; Barreira, LuísaTetraselmis striata CTP4 is a euryhaline, robust, fast-growing microalga suitable for wastewater treatment and industrial production. Lipid production was induced through a two-stage cultivation strategy: a 1st stage under standard growth-promoting conditions (100 mu mol photons m- 2 s- 1, salinity 36 ppt and 20 degrees C) to achieve high biomass concentration and a 2nd stage of 6 days for lipid induction by the application of abiotic stresses such as nutrient depletion, high light intensity (200 and 400 mu mol photons m- 2 s- 1), high salinity (75 and 100 ppt), and extreme temperatures (5 and 35 degrees C). Although nutrient depletion always resulted in a decrease in biomass productivity, it had also the highest impact on lipid induction. The highest lipid content (43.2%) and lipid productivity (29.2 mg L-1 d-1) were obtained using a combination of nutrient depletion and high light intensity (400 mu mol m- 2 s- 1). The fatty acid profile was mainly composed of C16:0 (palmitic), C18:1 (oleic) and C18:2 (linoleic) acids. The low content of unsaturated fatty acids and absence of C18:3 (linolenic) acid render the oil of this microalga suitable for biodiesel production, a renewable source of energy.
- Isolation of a euryhaline microalgal strain, Tetraselmis sp CTP4, as a robust feedstock for biodiesel productionPublication . Pereira, Hugo; Gangadhar, Katkam N.; Schulze, Peter S.C.; Santos, Tamara; de Sousa, Carolina Bruno; Schueler, Lisa; Custódio, Luísa; Malcata, F. Xavier; Gouveia, Luísa; Varela, J.; Barreira, LuísaBioprospecting for novel microalgal strains is key to improving the feasibility of microalgae-derived biodiesel production. Tetraselmis sp. CTP4 (Chlorophyta, Chlorodendrophyceae) was isolated using fluorescence activated cell sorting (FACS) in order to screen novel lipid-rich microalgae. CTP4 is a robust, euryhaline strain able to grow in seawater growth medium as well as in non-sterile urban wastewater. Because of its large cell size (9-22 mu m), CTP4 settles down after a six-hour sedimentation step. This leads to a medium removal efficiency of 80%, allowing a significant decrease of biomass dewatering costs. Using a two-stage system, a 3-fold increase in lipid content (up to 33% of DW) and a 2-fold enhancement in lipid productivity (up to 52.1 mg L-1 d(-1)) were observed upon exposure to nutrient depletion for 7 days. The biodiesel synthesized from the lipids of CTP4 contained high levels of oleic acid (25.67% of total fatty acids content) and minor amounts of polyunsaturated fatty acids with >= 4 double bonds (< 1%). As a result, this biofuel complies with most of the European (EN14214) and American (ASTM D6751) specifications, which commonly used microalgal feedstocks are usually unable to meet. In conclusion, Tetraselmis sp. CTP4 displays promising features as feedstock with lower downstream processing costs for biomass dewatering and biodiesel refining.
- Trends and strategies to enhance triacylglycerols and high-value compounds in microalgaePublication . Lisa Schueler, Lisa M. Schueler; Schulze, Peter S.C.; Pereira, Hugo; Barreira, Luísa; Leon, Rosa; J. C. or Varela J. or Varela J.C.S., VarelaMicroalgae are important sources of triacylglycerols (TAGs) and high-value compounds such as carotenoids and long-chain polyunsaturated fatty acids (LC-PUFAs). TAGs are feedstocks for biofuels or edible oils; carotenoids are used as pigments in the food and feed industries; and LC-PUFAs are beneficial for human health, being also key to the correct development of fish in aquaculture. Current trends in microalgal biotechnology propose the combined production of biofuels with high-value compounds to turn large-scale production of microalgal biomass into an economically feasible venture. As TAGs, carotenoids and LC-PUFAs are lipophilic biomolecules, they not only share biosynthetic precursors and storage sinks, but also their regulation often depends on common environmental stimuli. In general, stressful conditions favor carotenoid and TAGs biosynthesis, whereas the highest accumulation of LC-PUFAs is usually obtained under conditions promoting growth. However, there are known exceptions to these general rules, as a few species are able to accumulate LC-PUFAs under low light, low temperature or long-term stress conditions. Thus, future research on how microalgae sense, transduce and respond to environmental stress will be crucial to understand how the biosynthesis and storage of these lipophilic molecules are regulated. The use of high-throughput methods (e.g. fluorescent activated cell sorting) will provide an excellent opportunity to isolate triple-producers, i.e. microalgae able to accumulate high levels of LC-PUFAs, carotenoids and TAGs simultaneously. Comparative transcriptomics between wild type and tripleproducers could then be used to identify key gene products involved in the regulation of these biomolecules even in microalgal species not amenable to reverse genetics. This combined approach could be a major step towards a better understanding of the microalgal metabolism under different stress conditions. Moreover, the generation of triple-producers would be essential to raise the biomass value in a biorefinery setting and contribute to meet the world's rising demand for food, feed and energy.
- Improved production of lutein and β-carotene by thermal and light intensity upshifts in the marine microalga Tetraselmis sp. CTP4Publication . Schüler, Lisa Maylin; Santos, Tamara; Pereira, Hugo; Duarte, Paulo; Katkam, Dr. Gangadhar N.; Florindo, Claudia; Schulze, Peter S.C.; Barreira, Luísa; Varela, JoãoThe industrial microalga Tetraselmis sp. CTP4 is a promising candidate for aquaculture feed, novel food, cosmeceutical and nutraceutical due to its balanced biochemical profile. To further upgrade its biomass value, carotenogenesis was investigated by testing four environmental factors, namely temperature, light intensity, salinity and nutrient availability over different growth stages. The most important factor for carotenoid induction in this species is a sufficient supply of nitrates leading to an exponential growth of the cells. Furthermore, high temperatures of over 30 degrees C compared to lower temperatures (10 and 20 degrees C) induced the accumulation of carotenoids in this species. Remarkably, the two different branches of carotenoid synthesis were regulated depending on different light intensities. Contents of beta-carotene were 3-fold higher under low light intensities (33 mu mol m(-2) s(-1)) while lutein contents increased 1.5-fold under higher light intensities (170 and 280 mu mol m(-2) s(-1)). Nevertheless, highest contents of carotenoids (8.48 +/- 0.47 mg g(-1) DW) were found upon a thermal upshift from 20 degrees C to 35 degrees C after only two days at a light intensity of 170 mu mol m(-2) s(-1). Under these conditions, high contents of both lutein and beta-carotene were reached accounting for 3.17 +/- 0.18 and 3.21 +/- 0.18 mg g(-1) DW, respectively. This study indicates that Tetraselmis sp. CTP4 could be a sustainable source of lutein and beta-carotene at locations where a robust, euryhaline, thermotolerant microalgal strain is required.
- Drying microalgae using an industrial solar dryer: a biomass quality assessmentPublication . Schmid, Benjamin; Navalho, Sofia; Schulze, Peter S.C.; Van De Walle, Simon; Van Royen, Geert; Schüler, Lisa M.; Maia, Inês Beatriz; Bastos, Carolina; Baune, Marie-Christin; Januschewski, Edwin; Coelho, Ana; Pereira, Hugo; Varela, João; Navalho, João; Rodrigues, Alexandre Miguel CavacoMicroalgae are considered a promising resource of proteins, lipids, carbohydrates, and other functional biomolecules for food and feed markets. Competitive drying solutions are required to meet future demands for high-quality algal biomass while ensuring proper preservation at reduced costs. Since often used drying methods, such as freeze or spray drying, are energy and time consuming, more sustainable processes remain to be developed. This study tested an indirect and hybrid solar dryer as an alternative to conventional freeze drying of industrially produced Tetraselmis chui and Nannochloropsis oceanica wet paste. The effects of the drying method on biomass quality parameters, including biochemical profiles, functional properties, and microbial safety, were assessed. No significant differences were found between the applied drying technologies for total proteins, carbohydrates, lipids, and fatty acid profiles. On the other hand, some pigments showed significant differences, displaying up to 44.5% higher contents in freeze-dried samples. Minor differences were also registered in the mineral profiles (<10%). Analyses of microbial safety and functional properties of the solar-dried biomass appear adequate for food and feed products. In conclusion, industrial solar drying is a sustainable technology with a high potential to preserve high-quality microalgal biomass for various markets at expected lower costs.
- 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.
- 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.
- Carotenoid biosynthetic gene expression, pigment and n-3 fatty acid contents in carotenoid-rich Tetraselmis striata CTP4 strains under heat stress combined with high lightPublication . Schüler, Lisa Maylin; Bombo, Gabriel; Duarte, Paulo; Santos, Tamara; Maia, Inês Beatriz; Pinheiro, Filipa; Marques, José; Jacinto, Rita; Schulze, Peter S.C.; Pereira, Hugo; Barreira, Luísa; Varela, JoãoIn this study, two carotenoid-rich strains of the euryhaline microalga Tetraselmis striata CTP4 were isolated by random mutagenesis combined with selection via fluorescence activated cell sorting and growth on norflurazon. Both strains, ED5 and B11, showed an up to 1.5-fold increase in carotenoid contents as compared with the wildtype, independent of the growth conditions. More specifically, violaxanthin, beta-carotene and lutein contents reached as high as 1.63, 4.20 and 3.81 mg g-1 DW, respectively. Genes coding for phytoene synthase, phytoene desaturase, lycopene-beta-cyclase and epsilon-ring hydroxylase involved in carotenoid biosynthesis were found to be upregulated in ED5 and B11 cells as compared to the wildtype. Both strains showed higher contents of eicosapentaenoic acid as compared with those of the wildtype, reaching up to 4.41 and 2.88 mg g-1 DW, respectively. Overall, these results highlight the complexity of changes in carotenoid biosynthesis regulation that are required to improve pigment contents in microalgae.
- Flashing LEDs for microalgal productionPublication . Schulze, Peter S.C.; Guerra, Rui Manuel Farinha das Neves; Pereira, Hugo; Lisa Schueler, Lisa M. Schueler; J. C. or Varela J. or Varela J.C.S., VarelaFlashing lights are next-generation tools to mitigate light attenuation and increase the photosynthetic efficiency of microalgal cultivation systems illuminated by light-emitting diodes (LEDs). Optimal flashing light conditions depend on the reaction kinetics and properties of the linear electron transfer chain, energy dissipation, and storage mechanisms of a phototroph. In particular, extremely short and intense light flashes potentially mitigate light attenuation in photobioreactors without impairing photosynthesis. Intelligently controlling flashing light units and selecting electronic components can maximize light emission and energy efficiency. We discuss the biological, physical, and technical properties of flashing lights for algal production. We combine recent findings about photosynthetic pathways, self-shading in photobioreactors, and developments in solid-state technology towards the biotechnological application of LEDs to microalgal production.
- Dunaliella viridis TAV01: A halotolerant, Protein-Rich Microalga from the Algarve CoastPublication . Bombo, Gabriel; Cristofoli, N. L.; Santos, Tamara; Schüler, Lisa M.; Maia, Inês Beatriz; Pereira, Hugo; Barreira, Luísa; Varela, JoãoTolerance to harsh environmental conditions, high growth rates and an amino acid profile adequate for human consumption are beneficial features observed in Dunaliella viridis TAV01, a novel strain isolated from a salt pond in the Algarve, Portugal. TAV01 was identified down to the species level by maximum likelihood and Bayesian phylogenetic analyses of the ribosomal internal transcribed spacers one and two regions (ITS1 and ITS-2) and was supported by ITS2 secondary structure analysis. The biochemical profile revealed high protein (35.7 g 100 g−1 DW; 65% higher than the minimum recommended by the World Health Organization) and lipid contents (21.3 g 100 g−1 DW), a relatively higher proportion of the polyunsaturated fatty acids (PUFAs), α-linolenic (26.3% of total fatty acids (TFA)) and linoleic acids (22.8% of TFA), compared to those of other Dunaliella strains, and a balanced essential amino acids profile containing significant levels of leucine, phenylalanine, valine, and threonine. The major carotenoid was lutein, making up over 85% of total carotenoids. The presence of high-quality natural products in D. viridis TAV01 offers the possibility of using this new strain as a valuable biological resource for novel feed or food products as ingredients or supplements.