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- Effects of LED lighting on Nannochloropsis oceanica grown in outdoor raceway pondsPublication . Carneiro, M.; Maia, Inês Beatriz; Cunha, P.; Guerra, I.; Magina, T.; Santos, Tamara; Schulze, Peter S.C.; Pereira, H.; Malcata, F. X.; Navalho, J.; Silva, J.; Otero, A.; Varela, JoãoGrowth in most microalgal mass cultivation systems is light-limited, particularly in raceway ponds (RWP) where the light path is higher. Artificial lighting can be a promising solution to diminishing dark zones and enhance microalgal productivity. Therefore, our goal was to prevent the cell shift from photosynthesis to a respiration-only stage by resorting to LED illumination. Nannochloropsis oceanica cultures were accordingly grown out-doors in a preliminary small-scaleexperiment, followed by pilot-scale trials. In the former, three 3.0-m(2) RWP were set up under three distinct conditions: 1) without LEDs (control); 2) LEDs turned on during the night; and 3) LEDs turned on for 24 h. In the pilot-scale trial, one of two 28.9-m(2) pilot-scale RWPs was coupled to the best LED setup - determined in the small-scale preliminary experiment - using the same light intensity (normal mode) and half of the intensity (economy mode), with the second RWP serving as a control. In the preliminary experiment, the use of LEDs for 24 h was deemed as not helpful during daytime, before the culture reached asymptotic to 0.5 g DW L-1 - when dark zones appeared during the day due to sunlight attenuation in the 0.1 m-deep cultures. Overall, use of LEDs increased biomass growth chiefly by increasing nighttime productivities - materialized in higher chlorophyll, protein, and carbohydrate productivities in LED-lit cultures. A higher impact of LED lighting was observed under lower sunlight irradiances. A preliminary economic analysis indicates that use of LEDs in RWPs outdoors should be considered for high-value metabolites only.
- 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.
- Pilot scale production of Crocosphaera chwakensis CCY0110 and evaluation of its biomass nutritional potentialPublication . Matinha-Cardoso, Jorge; Santos, Tamara; Pereira, Hugo; Varela, João; Tamagnini, Paula; Mota, RitaIn recent years, the large-scale cultivation and commercialization of microalgae/cyanobacteria biomasses have become a worldwide trend, mainly directed to the animal and human nutrition markets due to their outstanding nutritional value. However, only a very limited number are currently classified as food ingredients by Food Safety regulators worldwide. Crocosphaera chwakensis CCY0110 is a marine unicellular cyanobacterium that produces a promising and versatile extracellular carbohydrate polymer (Cyanoflan). Therefore, envisaging a biorefinery approach with a multi-product stream (zero-waste), C. chwakensis biomass was for the first time cultivated at pilot-scale in a 120 L flat panel photobioreactor and its nutritional composition was evaluated. The results obtained revealed high protein and fat-soluble vitamins content (similar to 54 g of protein, 6 mg vitamin A and 25 mg vitamin E per 100 g biomass dry weight), coupled with a balanced amount of essential amino acids and n-3 polyunsaturated fatty acids (36 % of total fatty acids). Moreover, C. chwakensis biomass can also be considered as a great source of important minerals, such as potassium (3 g per 100 g DW), magnesium (0.5 g per 100 g DW) and iron (95 mg per 100 g DW), as well as phycocyanin, a high-value blue pigment with a wide array of applications. Overall, C. chwakensis biomass displays a nutritional composition that outdo traditional feedstocks and competes with already established and commercially available cyanobacteria and microalgae. This work highlights the potential of C. chwakensis as a multi-product cyanobacterium for blue bioeconomy, combining the production of a promising biopolymer with biomass valorization.
- 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.
- Influence of cultivation salinity in the nutritional composition, antioxidant capacity and microbial quality of Salicornia ramosissima commercially produced in soilless systemsPublication . Lima, Alexandre R.; Castaneda-Loaiza, Viana; Salazar, Miguel; Nunes, Carla; Quintas, Celia; Gama, Florinda; Pestana, Maribela; Correia, Pedro J.; Santos, Tamara; Varela, João; Barreira, Luísathe consumption of halophytes as healthy gourmet food has increased considerably in the past few years. However, knowledge on the nutritional profile of domesticated halophytes is scarce and little is known on which cultivation conditions can produce plants with the best nutritional and functional properties. In this context, Salicornia ramosissima J. Woods was cultivated in six different salt concentrations, ranging from 35 to 465 mM of NaCl. Both the nutritional profile, the antioxidant capacity, and microbial quality of the produced plants were evaluated including minerals and vitamins. Salt has a marked effect on growth, which decreases for salinities higher than 110 mM. Nonetheless, plants cultivated with intermediate levels of salinity (110 and 200 mM) revealed better antioxidant status with higher amounts of phenolic compounds. Overall, results from this paper indicated that soilless culture systems using low-intermediate salinities produces S. ramosissima plants fit for commercialization and human consumption.
- 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.
- 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.
- The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish modelsPublication . Carletti, Alessio; Rosa, Joana; Pes, Katia; Borges, Inês; Santos, Tamara; Barreira, Luísa; Varela, João; Pereira, Hugo; Cancela, M. Leonor; J. Gavaia, Paulo; Laizé, VincentSkeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.
- Incorporation of defatted microalgal biomass (Tetraselmis sp. CTP4) at the expense of soybean meal as a feed ingredient for juvenile gilthead seabream (Sparus aurata)Publication . Pereira, Hugo; Sardinha, Manuel; Santos, Tamara; Gouveia, Luisa; Barreira, Luísa; Dias, Jorge; Varela, JoãoThe forecasted growth of the aquaculture sector requires the use of novel and sustainable ingredients in aquaculture feeds. A study was undertaken to evaluate the effect of a 10% incorporation of defatted microalgal biomass (DMB) of Tetraselmis sp. CTP4, used at the expense of dehulled solvent-extracted soybean meal (SBM), on the growth performance, nutrient digestibility and physiological response to confinement stress in gilthead seabream juveniles. The trial comprised two dietary treatments: a control diet (CTRL) with relatively high levels of marine-derived proteins and 10% SBM; and a test diet (DMB10) with the incorporation of 10% DMB at the expense of SBM, while maintaining a fair constancy of all other ingredients. Triplicate groups of 30 fish, with a mean initial body weight of 6.0 +/- 0.2 g were fed the experimental diets for 61 days. At the end of the trial, fish tripled their initial body weight, but the overall growth performance criteria (final body weight, daily growth index, feed conversion ratio and protein efficiency ratio), whole-body composition and nutrient retention were not significantly affected by the dietary treatments (p > 0.05). The DMB10 diet showed a significantly higher apparent digestibility coefficients (ADC) of dry matter, energy and phosphorus (p < 0.05). When measured as an isolated feed ingredient, the DMB had an ADC of protein, fat, energy and phosphorus of 87.9, 85.3, 75.5 and 41.4%, respectively. After an acute confinement stress test, fish fed with DMB10 diet displayed a significantly lower plasma cortisol response (120 +/- 23 ng/mL) than those fed with the control diet (160 +/- 33 ng/mL) (p < 0.05). Overall results showed that DMB, issued from biorefinery processes, could potentially spare the use of soybean meal in aquaculture feeds, contributing towards a reduction of the current protein deficit in the European market.
- Operation regimes: A comparison based on Nannochloropsis oceanica biomass and lipid productivityPublication . Guerra, Inês; Pereira, Hugo; Costa, Margarida; Silva, Joana T.; Santos, Tamara; Varela, João; Mateus, Marília; Silva, JoanaMicroalgae are currently considered to be a promising feedstock for biodiesel production. However, significant research efforts are crucial to improve the current biomass and lipid productivities under real outdoor production conditions. In this context, batch, continuous and semi-continuous operation regimes were compared during the Spring/Summer seasons in 2.6 m(3) tubular photobioreactors to select the most suitable one for the production of the oleaginous microalga Nannochloropsis oceanica. Results obtained revealed that N. oceanica grown using the semi-continuous and continuous operation regimes enabled a 1.5-fold increase in biomass volumetric productivity compared to that cultivated in batch. The lipid productivity was 1.7-fold higher under semi-continuous cultivation than that under a batch operation regime. On the other hand, the semi-continuous and continuous operation regimes spent nearly the double amount of water compared to that of the batch regime. Interestingly, the biochemical profile of produced biomass using the different operation regimes was not affected regarding the contents of proteins, lipids and fatty acids. Overall, these results show that the semi-continuous operation regime is more suitable for the outdoor production of N. oceanica, significantly improving the biomass and lipid productivities at large-scale, which is a crucial factor for biodiesel production.