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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.
- Optimisation of biomass production and nutritional value of two marine diatoms (Bacillariophyceae), Skeletonema costatum and Chaetoceros calcitransPublication . Bastos, Carolina R. V.; Maia, Inês Beatriz; Pereira, Hugo; Navalho, João; Varela, JoãoS. costatum and C. calcitrans are two cosmopolitan high-value centric diatoms, with a rich nutritional profile. The following work optimised the culture medium of S. costatum and C. calcitrans cultures, respectively, in a stepwise process as follows: 2.4 mM and 1.2 mM of silicate, 4 mM of nitrate, 100 µM of phosphate, 20 and 80 µM iron, and 0.5 mL L−1 of micronutrients. The results that were obtained revealed an increase in biomass productivity with a 1.8- and 3.2-fold increase in biomass that was produced by S. costatum and C. calcitrans, respectively. The biochemical profile showed an increase in high-value PUFAs such as 2.6-fold and 2.3-fold increase in EPA for S. costatum and C. calcitrans, respectively, whilst a 2.6-fold increase in DHA was detected in S. costatum cultures. The present work provides the basic tools for the industrial cultivation of S. costatum and C. calcitrans with enhanced productivity as well as improved biomass quality, two factors which are highly relevant for a more effective application of these diatoms to aquaculture and nutraceutical production.
- Antileishmanial activity of meroditerpenoids from the macroalgae Cystoseira baccataPublication . Sousa, Carolina Bruno de; Gangadhar, Katkam N.; Morais, Thiago R.; Conserva, Geanne A. A.; Vizetto-Duarte, C; Pereira, H.; Laurenti, Marcia D.; Campino, Lenea; Levy, Debora; Uemi, Miriam; Barreira, Luísa; Custódio, L.; Passero, Luiz Felipe D.; Lago, Joao Henrique G.; Varela, JoãoThe development of novel drugs for the treatment of leishmaniases continues to be crucial to overcome the severe impacts of these diseases on human and animal health. Several bioactivities have been described in extracts from macroalgae belonging to the Cystoseira genus. However, none of the studies has reported the chemical compounds responsible for the antileishmanial activity observed upon incubation of the parasite with the aforementioned extracts. Thus, this work aimed to isolate and characterize the molecules present in a hexane extract of Cystoseira baccata that was found to be bioactive against Leishmania infantum in a previous screening effort. A bioactivity-guided fractionation of the C. baccata extract was carried out and the inhibitory potential of the isolated compounds was evaluated via the MIT assay against promastigotes and murine macrophages as well as direct counting against intracellular amastigotes. Moreover, the promastigote ultrastructure, DNA fragmentation and changes in the mitochondrial potential were assessed to unravel their mechanism of action. In this process, two antileishmanial meroditerpenoids, (3R)- and (3S)-tetraprenyltoluquinol (1a/1b) and (3R)- and (3S)-tetraprenyltoluquinone (2a/2b), were isolated. Compounds 1 and 2 inhibited the growth of the L. infantum promastigotes (IC50 = 44.9 +/- 4.3 and 94.4 +/- 10.1 mu M, respectively), inducing cytoplasmic vacuolization and the presence of coiled multilamellar structures in mitochondria as well as an intense disruption of the mitochondrial membrane potential. Compound 1 decreased the intracellular infection index (IC50 = 25.0 +/- 4.1 mu M), while compound 2 eliminated 50% of the intracellular amastigotes at a concentration > 88.0 mu M. This work identified compound 2 as a novel metabolite and compound 1 as a biochemical isolated from Cystoseira algae displaying antileishmanial activity. Compound 1 can thus be an interesting scaffold for the development of novel chemotherapeutic molecules for canine and human visceral leishmaniases studies. This work reinforces the evidence of the marine environment as source of novel molecules. (C) 2017 Elsevier Inc. All rights reserved.
- 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.
- Assessment and comparison of the properties of biodiesel synthesized from three different types of wet microalgal biomassPublication . Gangadhar, Katkam N.; Pereira, Hugo; Diogo, Herminio P.; Borges dos Santos, R. M.; Devi, B. L. A. Prabhavathi; Prasad, R. B. N.; Custódio, Luísa; Xavier Malcata, F.; Varela, João; Barreira, LuísaIn recent years, microalgae-based carbon-neutral biofuels (i.e., biodiesel) have gained considerable interest due to high growth rate and higher lipid productivity of microalgae during the whole year, delivering continuous biomass production as compared to vegetable-based feedstocks. Therefore, biodiesel was synthesized from three different microalgal species, namely Tetraselmis sp. (Chlorophyta) and Nannochloropsis oculata and Phaeodactylum tricornutum (Heterokontophyta), and the fuel properties of the biodiesel were analytically determined, unlike most studies which rely on estimates based on the lipid profile of the microalgae. These include density, kinematic viscosity, total and free glycerol, and high heating value (HHV), while cetane number (CN) and cold filter plugging point (CFPP) were estimated based on the fatty acid methyl ester profile of the biodiesel samples instead of the lipid profile of the microalgae. Most biodiesel properties abide by the ASTM D6751 and the EN 14214 specifications, although none of the biodiesel samples met the minimum CN or the maximum content of polyunsaturated fatty acids with a parts per thousand yen4 double bonds as required by the EN 14214 reference value. On the other hand, bomb calorimetric experiments revealed that the heat of combustion of all samples was on the upper limit expected for biodiesel fuels, actually being close to that of petrodiesel. Post-production processing may overcome the aforementioned limitations, enabling the production of biodiesel with high HHV obtained from lipids present in these microalgae.
- 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.
- Isolation and fatty acid profile of selected microalgae strains from the Red Sea for biofuel productionPublication . Pereira, Hugo; Barreira, Luisa; Custodio, Luisa; Alrokayan, Salman; Mouffouk, Fouzi; Varela, Joao; Abu-Salah, Khalid M.; Ben-Hamadou, RadhouanThe isolation of lipid-rich autochthonous strains of microalgae is a crucial stage for the development of a microalgae-based biofuel production plant, as these microalgae already have the necessary adaptations to withstand competition, predation and the temperatures observed at each production site. This is particularly important in extreme climates such as in Saudi Arabia. Resorting to fluorescence activated cell sorting (FACS) we screened for and isolated several microalgal strains from samples collected from the Red Sea. Relying on the fluorescence of BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diazasindacene) and growth performance, four promising candidates were identified and the total lipid content and fatty acid profile was assessed for biofuels production. Selected isolates were classified as chlorophytes, belonging to three different genera: Picochlorum, Nannochloris and Desmochloris. The lipid contents were assessed microscopically by means of BODIPY 505/515-associated fluorescence to detect intracellular lipid bodies, which revealed several lipid drops in all selected strains. This result was confirmed by lipid gravimetric determination, which demonstrated that all strains under study presented inner cell lipid contents ranging from 20% to 25% of the biomass dry weight. Furthermore, the fatty acid methyl esters profile of all strains seems ideal for biodiesel production due to a low degree of polyunsaturated fatty acid methyl esters and high amount of palmitic and oleic acids.
- 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.
- Industrial production of Phaeodactylum tricornutum for CO2 mitigation: biomass productivity and photosynthetic efficiency using photobioreactors of different volumesPublication . Quelhas, Pedro M.; Trovao, Mafalda; Silva, Joana T.; Machado, Adriana; Santos, Tamara; Pereira, Hugo; Varela, João; Simoes, Manuel; Silva, Joana L.The photosynthetic efficiency (PE) and potential of Phaeodactylum tricornutum for CO2 mitigation in industrial tubular photobioreactors (PBRs) of different volumes were evaluated. A preliminary assay was performed at lab-scale to optimize the salt concentration of the culture medium. Interestingly, salinity did not affect the growth of P. tricornutum at concentrations of 2.5, 5, 10, and 20 g L-1. Higher volumetric productivities were achieved in the 2.5-m(3) tubular PBR (0.235 g L-1 day(-1)), followed by 35- and 10-m(3) PBRs. Maximum areal productivities corresponded to 48.5, 45.0, and 12.8 g m(-2) day(-1) for the 35-, 10-, and 2.5-m(3) PBRs, respectively. PE was thus higher in the 35- and 10-m(3) PBRs (2.21 and 2.08%, respectively). The 10- and 35-m(3) PBR showed CO2 mitigation efficiencies of 60 and 41%, respectively, of the CO2 introduced into the PBR, corresponding to 2.3 and 2.5 g of fixed CO2 per g of biomass. Overall, cultivation of P. tricornutum couples high PE and areal productivity when the industrial PBRs were used, particularly PBRs of larger volumes. This improved PE performance with larger PBR volumes strongly suggests that large-scale cultivation of this diatom holds great potential for industrial CO2 mitigation.