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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.
- Nutritional and functional evaluation of inula crithmoides and mesembryanthemum nodiflorum grown in different salinities for human consumptionPublication . Ribeiro Lima, Alexandre; Gama, Florinda; Castañeda-Loaiza, Viana; Costa, Camila; Schueler, Lisa M.; Santos, Tamara; Salazar, Miguel; Nunes, Carla; Cruz, Rui M. S.; Varela, J.; Barreira, LuísaThe nutritional composition and productivity of halophytes is strongly related to the biotic/abiotic stress to which these extremophile salt tolerant plants are subjected during their cultivation cycle. In this study, two commercial halophyte species (Inula crithmoides and Mesembryanthemum nodiflorum) were cultivated at six levels of salinity using a soilless cultivation system. In this way, it was possible to understand the response mechanisms of these halophytes to salt stress. The relative productivity decreased from the salinities of 110 and 200 mmol L−1 upwards for I. crithmoides and M. nodiflorum, respectively. Nonetheless, the nutritional profile for human consumption remained balanced. In general, I. crithmoides vitamin (B1 and B6) contents were significantly higher than those of M. nodiflorum. For both species, β-carotene and lutein were induced by salinity, possibly as a response to oxidative stress. Phenolic compounds were more abundant in plants cultivated at lower salinities, while the antioxidant activity increased as a response to salt stress. Sensory characteristics were evaluated by a panel of culinary chefs showing a preference for plants grown at the salt concentration of 350 mmol L−1. In summary, salinity stress was effective in boosting important nutritional components in these species, and the soilless system promotes the sustainable and safe production of halophyte plants for human consumption.
- 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.
- Wild vs cultivated halophytes: Nutritional and functional differencesPublication . Castañeda-Loaiza, Viana; Oliveira, Marta; Santos, Tamara; Schüler, Lisa M.; Ribeiro Lima, Alexandre; Gama, Florinda; Salazar, Miguel; Neng, N.R.; Nogueira, J. M. F.; Varela, João; Barreira, LuísaSome halophyte plants are currently used in gourmet cuisine due to their unique organoleptic properties. Moreover, they exhibit excellent nutritional and functional properties, being rich in polyphenolics and vitamins. These compounds are associated to strong antioxidant activity and enhanced health benefits. This work compared the nutritional properties and antioxidant potential of three species (Mesembryanthemum nodiflorum, Suaeda maritima and Sarcocornia fruticosa) collected in saltmarshes from Portugal and Spain with those of cultivated plants. the latter were generally more succulent and had higher contents of minerals than plants obtained from the wild and contained less fibre. All species assayed are a good source of proteins, fibres and minerals. Additionally, they are good sources of carotenoids and vitamins A, C and B-6 and showed good antioxidant potential particularly S. maritima. Chromatographic analysis of the phenolic profile revealed that ferulic and caffeic acids as the most relevant phenolic compounds detected in the halophytes tested.
- Improvement of carotenoid extraction from a recently isolated, robust microalga, Tetraselmis sp. CTP4 (chlorophyta)Publication . Lisa Schueler, Lisa M. Schueler; Katkam, Dr. Gangadhar N.; Duarte, Paulo A. S.; Placines, Chloé; Molina-Márquez, Ana María; Léon-Bañares, Rosa; Sousa, Vânia S.; Varela, João; Barreira, LuísaIn recent years, there has been increasing consumer interest in carotenoids, particularly of marine sustainable origin with applications in the food, cosmeceutical, nutritional supplement and pharmaceutical industries. For instance, microalgae belonging to the genus Tetraselmis are known for their biotechnologically relevant carotenoid profile. The recently isolated marine microalgal strain Tetraselmis sp. CTP4 is a fast-growing, robust industrial strain, which has successfully been produced in 100-m3 photobioreactors. However, there are no reports on total carotenoid contents from this strain belonging to T. striata/convolutae clade. Although there are several reports on extraction methods targeting chlorophytes, extraction depends on the strength of cell coverings, solvent polarity and the nature of the targeted carotenoids. Therefore, this article evaluates different extraction methods targeting Tetraselmis sp. CTP4, a strain known to contain a mechanically resistant theca. Here, we propose a factorial experimental design to compare extraction of total carotenoids from wet and freeze-dried microalgal biomass using four different solvents (acetone, ethanol, methanol or tetrahydrofuran) in combination with two types of mechanical cell disruption (glass beads or dispersion). The extraction efficiency of the methods was assessed by pigment contents and profiles present in the extracts. Extraction of wet biomass by means of glass bead-assisted cell disruption using tetrahydrofuran yielded the highest amounts of lutein and β-carotene (622 ± 40 and 618 ± 32 µg g-1 DW, respectively). Although acetone was slightly less efficient than tetrahydrofuran, it is preferable due to its lower costs and toxicity.
- Biomass valorization via pyrolysis in microalgae-based wastewater treatment: challenges and opportunities for a circular bioeconomyPublication . de Morais, Etiele Greque; da Silveira, Jéssica Teixeira; Schüler, Lisa Maylin; de Freitas, Barbara Catarina Bastos; Costa, Jorge Alberto Vieira; de Morais, Michele Greque; Ferrer, Ivet; Barreira, LuísaMicroalgae-based wastewater treatment technology is a sustainable and environmentally friendly alternative to conventional treatment systems. The biomass produced during microalgae-based wastewater treatment can be valorized via pyrolysis to generate multiple valuable products, such as biochar, bio-oil, and pyrolytic gas. This study summarizes the potential of pyrolysis for valorizing microalgal biomass produced from wastewater treatment. It shows how pyrolysis can provide a variety of valuable products, the composition of which is influenced by the type of microalgae used, the operating conditions of the pyrolysis process, and the presence of contaminants in the biomass. It also highlights the main challenges to be addressed before pyrolysis can be adopted to valorize microalgae biomass. These challenges include the high energy requirements of pyrolysis, the need for further research to optimize the process, and the potential for pyrolysis to produce harmful emissions. Despite this, pyrolysis appears as a promising technology with potential to contribute to the sustainable development of a circular economy. Future research should address these challenges and develop more efficient and environmentally friendly pyrolysis processes.