Optimization of Emiliania huxleyi growth for production of n-3 polyunsaturated fatty acids and novel compounds with osteogenic activity

Emiliania huxleyi é uma das mais abundantes espécies de cocolitoforídeos (Haptophyta) e é responsável por vastos blooms em todo o mundo, sendo até visíveis do espaço. A abundância de E. huxleyi nos oceanos sugere fortemente que é uma microalga promissora para a produção industrial de larga escala, com possíveis aplicações biotecnológicas. A sua capacidade de calcificação, devido à produção de placas de calcite, sugere ainda que poderá ser usada para estudos de acidificação dos oceanos, com potencial para mitigação de CO2. E. huxleyi é um organismo unicelular com um reduzido tamanho (4-6 μm) que produz cocólitos em vesículas intracelulares especializadas. Possui também um ciclo de vida haplo-diplonte complexo, com 3 tipos celulares diferentes: células portadoras de cocólitos (células C diploides), células nuas não-móveis (células N) e células móveis portadores de escamas (células S haploides). Os diferentes tipos celulares podem ser induzidos durante o ciclo de vida, levando à diferenciação celular. O ciclo de vida haplo-diplonte tem também um papel muito importante para a sobrevivência da espécie. Esta espécie possui várias caraterísticas com interesse biotecnológico, devido à síntese de ácidos gordos polinsaturados de elevado valor comercial e à produção de pigmentos que poderão servir como substitutos de colorantes artificiais. Além disso, E. huxleyi possui ainda uma caraterística diferente de outras espécies de microalgas mais produzidas industrialmente, que é a produção de cocólitos formados por CaCO3. Estes cocólitos demonstram também grande potencial, com aplicações em nanotecnologia ou ainda como substitutos à calcite industrial. A produção de CaCO3 sugere ainda a presença de compostos com atividade osteogénica. Sete estirpes de E. huxleyi foram adquiridas, procedendo-se ao seu crescimento sob condições controladas. Das sete estirpes, a estirpe RCC1250 foi a selecionada, uma vez que foi a que respondeu melhor às condições de scale-up. Fatores abióticos importantes – meio de cultura, temperatura e intensidade luminosa – foram otimizados para a estirpe E. huxleyi RCC1250 através do uso de fotobiorreatores de escala laboratorial Algem®. Cada ensaio teve a duração de 11 dias, com a monitorização das culturas a cada 2 dias, que incluía: contagens celulares, fluorometria, determinação da concentração de NO3- e observações microscópicas. O desempenho de crescimento foi superior usando Nutribloom® como meio de cultura, quando comparado com o crescimento observado com o meio de cultura K/2, que é considerado o meio de cultura “standard” para esta espécie. A concentração de NO3- também mostrou ser crucial para o crescimento, em que o meio de cultura a uma concentração de NO3- de 0.6 mM demonstrou ser inibitório, numa fase inicial do crescimento. Com este ensaio, ficou definido que o meio de cultura Nutribloom® a uma concentração de 0.3 mM de NO3- seria adicionado no dia 0 e, quando a cultura atingisse uma densidade ótica de 1, esta concentração seria aumentada para 0.6 mM, com adição de meio de cultura a cada 2 dias. A temperatura ótima de crescimento correspondeu a 23 ºC, mas também houve crescimento aos 26 ºC, mostrando um certo nível de adaptação a temperaturas mais altas, o que será vantajoso para uma produção industrial no sul de Portugal. Determinou-se também que 900 μmol fotões/m2/s corresponde à intensidade luminosa ótima em termos de densidade de fluxo de fotões. Foi ainda possível verificar a inibição da fotossíntese em culturas expostas a intensidades luminosas superiores a 1000 μmol fotões/m2/s. As condições “standard” (meio de cultura K/2 a uma concentração de NO3- de 0.3 mM, posteriormente aumentada para 0.6 mM, 17 ºC e 1219 μmol fotões/m2/s) foram também comparadas com as condições otimizadas (meio de cultura Nutribloom® a uma concentração de NO3- de 0.3 mM, posteriormente aumentada para 0.6 mM, 23 ºC e 900 μmol fotões/m2/s), resultando num aumento de crescimento significativo. No final dos ensaios nos fotobiorreatores Algem®, a biomassa foi recolhida por centrifugação e liofilizada para a determinação da composição bioquímica de E. huxleyi. Determinou-se também o conteúdo proteico e de pigmentos, a percentagem de lípidos totais e o perfil de ácidos gordos. A biomassa produzida sob as condições standard continha elevadas quantidades de ácidos gordos saturados e monoinsaturados, nomeadamente os ácidos gordos mirístico, palmítico e oleico. No entanto, a biomassa produzida sob as condições otimizadas continha elevadas quantidades de ácidos gordos polinsaturados (PUFA), nomeadamente os ácidos octadecatetrenóico (OTA) e docosahexenóico (DHA), que são conhecidos pelo seu elevado valor comercial. Aumentaram ainda a produção dos PUFAs anteriores em 4 e 5 vezes, respetivamente. O conteúdo proteico foi também significativamente superior nas culturas expostas às condições otimizadas. Elevadas quantidades de 19’-hexanoilofucoxantina e fucoxantina foram também obtidas sob condições otimizadas, com um aumento de 3 e 2 vezes, respetivamente. O potencial osteogénico de vários extratos de E. huxleyi (etanol, acetato de etilo e água) foi avaliado em larvas de peixe-zebra (Danio rerio) com 3 dias pós-fertilização, expostas por 3 dias a várias concentrações de cada extrato. Os extratos testados não afetaram a área da cabeça das larvas, sendo este o parâmetro usado para a correção da área do opérculo. Análises morfométricas das larvas coloradas com alizarin-red revelaram que o extrato etanólico a 10 μg/mL e 1 μg/mL aumentaram, respetivamente, a área do opérculo em 20 e 11% sobre o controlo (o primeiro tão alto quanto o controlo positivo). A aplicação do extrato de acetato de etilo também levou a um aumento do opérculo em 12% a 100 μg/mL, enquanto o extrato de água não demonstrou nenhum efeito significativo no crescimento do osso. Este ensaio mostrou a presença de compostos pro-osteogénicos, com potencial para desenvolvimento de um novo fármaco. Em conclusão, o presente estudo revelou uma nova perspetiva no impacto dos fatores abióticos no crescimento de E. huxleyi RCC1250. O meio de cultura e a sua concentração de NO3- demonstrou ter um papel fundamental no crescimento desta estirpe, assim como a temperatura e a intensidade luminosa. A otimização destes parâmetros levou também a um aumento significativo na produção de compostos de elevado valor comercial, como PUFAs n-3, fucoxantina e 19’-hexanoilofucoxantina. Deste modo, este trabalho não só permitiu o estabelecimento de um novo protocolo para o melhoramento do crescimento de E. huxleyi, mas também mostrou o seu potencial como uma fonte de compostos de elevado valor comercial e de importantes metabolitos secundários com atividade osteogénica na biomassa produzida.Emiliania huxleyi is one of the most abundant species of coccolithophores (Haptophyta) and is responsible for extensive blooms worldwide. The widespread abundance of E. huxleyi suggests that it may be a promising species for industrial production with high potential for biotechnological applications. Important abiotic factors – culture media, temperature and light intensity – were optimized for E. huxleyi RCC1250 using lab-scale Algem® photobioreactors. Growth performance was higher using Nutribloom® as culture medium as compared to K/2, which is the considered to be the “standard” medium for this species. Optimal temperature and light intensity were, respectively, 23ºC and 900 μmol photons/m2/s in Nutribloom® growth medium. The biomass produced contained high amounts of polyunsaturated fatty acids (PUFA), in particular octadecatetraenoic (OTA) and docosahexaenoic acids (DHA), which are known to have high market value. Optimized conditions increased the production of these PUFAs by 5- and 4-fold, respectively. High amounts of 19’-hexanoyloxyfucoxanthin and fucoxanthin were also achieved under optimized conditions with an increase of 2- and 3-fold. The osteogenic potential of several E. huxleyi extracts (i.e., ethanol, ethyl acetate and water) was assessed in zebrafish (Danio rerio) larvae at 3 days post-fertilization exposed for 3 days to a range of concentrations of each extract. Morphometric analysis of alizarin red-stained larvae revealed that the ethanolic extract at 10 μg/mL and 1 μg/mL increased, respectively, the operculum area by 20 and 11% over the control (the former as high as the positive control). Ethyl acetate extract also induced an operculum increase of 12% at 100 μg/mL, whereas water extract did not show any significant effect on bone growth. In conclusion, this work has not only established a new protocol to improve E. huxleyi growth performance but has shown the presence of high-value compounds and important secondary metabolites with osteogenic activity in the produced biomass

Optimisation of biomass production and nutritional value of two marine diatoms (Bacillariophyceae), Skeletonema costatum and Chaetoceros calcitrans

S. 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.info:eu-repo/semantics/publishedVersio

Dunaliella viridis TAV01: A halotolerant, Protein-Rich Microalga from the Algarve Coast

Tolerance 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.info:eu-repo/semantics/publishedVersio

Microalgal systems for wastewater treatment: technological trends and challenges towards waste recovery

Wastewater (WW) treatment using microalgae has become a growing trend due the economic and environmental benefits of the process. As microalgae need CO2, nitrogen, and phosphorus to grow, they remove these potential pollutants from wastewaters, making them able to replace energetically expensive treatment steps in conventional WW treatment. Unlike traditional sludge, biomass can be used to produce biofuels, biofertilizers, high value chemicals, and even next-generation growth media for “organically” grown microalgal biomass targeting zero-waste policies and contributing to a more sustainable circular bioeconomy. The main challenge in this technology is the techno-economic feasibility of the system. Alternatives such as the isolation of novel strains, the use of native consortia, and the design of new bioreactors have been studied to overcome this and aid the scale-up of microalgal systems. This review focuses on the treatment of urban, industrial, and agricultural wastewaters by microalgae and their ability to not only remove, but also promote the reuse, of those pollutants. Opportunities and future prospects are discussed, including the upgrading of the produced biomass into valuable compounds, mainly biofuels.This research was funded by the Foundation for Science and Technology (FCT) through UIDB/04326/2020 and the GreenTreat (PTDC/BTA-BTA/31567/2017) and Red CYTED P319RT0025— RENUWAL—Red Iberoamericana para el Tratamiento de Efluentes con Microalgas projects and CRESC-Algarve and the European Regional Development Fund (ERDF) programs via the ALGAVALOR (ALG-01-0247-FEDER-035234) project.info:eu-repo/semantics/publishedVersio

Potential of Coccolithophore microalgae as fillers in starch-based films for active and sustainable food packaging

Coccolithophore microalgae, such as Emiliania huxleyi (EHUX) and Chrysotila pseudoroscoffensis (CP), are composed of calcium carbonate (CaCO3) and contain bioactive compounds that can be explored to produce sustainable food packaging. In this study, for the first time, these microalgae were incorporated as fillers in starch-based films, envisioning the development of biodegradable and bioactive materials for food packaging applications. The films were obtained by solvent casting using different proportions of the filler (2.5, 5, 10, and 20%, w/w). For comparison, commercial CaCO3, used as filler in the plastic industry, was also tested. The incorporation of CaCO3 and microalgae (EHUX or CP) made the films significantly less rigid, decreasing Young’s modulus up to 4.7-fold. Moreover, the incorporation of microalgae hydrophobic compounds as lipids turned the surface hydrophobic (water contact angles > 90°). Contrary to what was observed with commercial CaCO3, the films prepared with microalgae exhibited antioxidant activity, increasing from 0.9% (control) up to 60.4% (EHUX 20%) of ABTS radical inhibition. Overall, the introduction of microalgae biomass improved hydrophobicity and antioxidant capacity of starch-based films. These findings should be considered for further research using coccolithophores to produce active and sustainable food packaging material.info:eu-repo/semantics/publishedVersio

The polar lipidome of cultured Emiliania huxleyi: a source of bioactive lipids with relevance for biotechnological applications

Polar lipids from microalgae have aroused greater interest as a natural source of omega-3 (n-3) polyunsaturated fatty acids (PUFA), an alternative to fish, but also as bioactive compounds with multiple applications. The present study aims to characterize the polar lipid profile of cultured microalga Emiliania huxleyi using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-MS) and fatty acids (FA) analysis by gas chromatography (GC-MS). The lipidome of E. huxleyi revealed the presence of distinct n-3 PUFA (40% of total FA), namely docosahexaenoic acid (22:6n-3) and stearidonic acid (18:4n-3), which give this microalga an increased commercial value as a source of n-3 PUFA present in the form of polar lipids. A total of 134 species of polar lipids were identified and some of these species, particularly glycolipids, have already been reported for their bioactive properties. Among betaine lipids, the diacylglyceryl carboxyhydroxymethylcholine (DGCC) class is the least reported in microalgae. For the first time, monomethylphosphatidylethanolamine (MMPE) has been found in the lipidome of E. huxleyi. Overall, this study highlights the potential of E. huxleyi as a sustainable source of high-value polar lipids that can be exploited for different applications, namely human and animal nutrition, cosmetics, and pharmaceuticals.FCT/ FCT/MCTES (Portugal) for the financial support to CICECO (UIDB/50011/2020 and 50011/2020), QOPNA (UID/QUI/00062/2019), LAQV-REQUIMTE (UIDB/50006/2020), CESAM (UIDB/50017/2020 and UIDP/50017/2020), RNEM (LISBOA-01-0145-FEDER-402-022125) and CCMAR: (UIDB/04326/2020).info:eu-repo/semantics/publishedVersio

Effects of LED lighting on Nannochloropsis oceanica grown in outdoor raceway ponds

Growth 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.info:eu-repo/semantics/publishedVersio

Drying microalgae using an industrial solar dryer: a biomass quality assessment

Microalgae 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.info:eu-repo/semantics/publishedVersio

Estratégias para o cuidado da pessoa com doença crônica : diabetes mellitus

Telemedicin

Global, regional, and national under-5 mortality, adult mortality, age-specific mortality, and life expectancy, 1970–2016: a systematic analysis for the Global Burden of Disease Study 2016

BACKGROUND: Detailed assessments of mortality patterns, particularly age-specific mortality, represent a crucial input that enables health systems to target interventions to specific populations. Understanding how all-cause mortality has changed with respect to development status can identify exemplars for best practice. To accomplish this, the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016) estimated age-specific and sex-specific all-cause mortality between 1970 and 2016 for 195 countries and territories and at the subnational level for the five countries with a population greater than 200 million in 2016. METHODS: We have evaluated how well civil registration systems captured deaths using a set of demographic methods called death distribution methods for adults and from consideration of survey and census data for children younger than 5 years. We generated an overall assessment of completeness of registration of deaths by dividing registered deaths in each location-year by our estimate of all-age deaths generated from our overall estimation process. For 163 locations, including subnational units in countries with a population greater than 200 million with complete vital registration (VR) systems, our estimates were largely driven by the observed data, with corrections for small fluctuations in numbers and estimation for recent years where there were lags in data reporting (lags were variable by location, generally between 1 year and 6 years). For other locations, we took advantage of different data sources available to measure under-5 mortality rates (U5MR) using complete birth histories, summary birth histories, and incomplete VR with adjustments; we measured adult mortality rate (the probability of death in individuals aged 15-60 years) using adjusted incomplete VR, sibling histories, and household death recall. We used the U5MR and adult mortality rate, together with crude death rate due to HIV in the GBD model life table system, to estimate age-specific and sex-specific death rates for each location-year. Using various international databases, we identified fatal discontinuities, which we defined as increases in the death rate of more than one death per million, resulting from conflict and terrorism, natural disasters, major transport or technological accidents, and a subset of epidemic infectious diseases; these were added to estimates in the relevant years. In 47 countries with an identified peak adult prevalence for HIV/AIDS of more than 0·5% and where VR systems were less than 65% complete, we informed our estimates of age-sex-specific mortality using the Estimation and Projection Package (EPP)-Spectrum model fitted to national HIV/AIDS prevalence surveys and antenatal clinic serosurveillance systems. We estimated stillbirths, early neonatal, late neonatal, and childhood mortality using both survey and VR data in spatiotemporal Gaussian process regression models. We estimated abridged life tables for all location-years using age-specific death rates. We grouped locations into development quintiles based on the Socio-demographic Index (SDI) and analysed mortality trends by quintile. Using spline regression, we estimated the expected mortality rate for each age-sex group as a function of SDI. We identified countries with higher life expectancy than expected by comparing observed life expectancy to anticipated life expectancy on the basis of development status alone. FINDINGS: Completeness in the registration of deaths increased from 28% in 1970 to a peak of 45% in 2013; completeness was lower after 2013 because of lags in reporting. Total deaths in children younger than 5 years decreased from 1970 to 2016, and slower decreases occurred at ages 5-24 years. By contrast, numbers of adult deaths increased in each 5-year age bracket above the age of 25 years. The distribution of annualised rates of change in age-specific mortality rate differed over the period 2000 to 2016 compared with earlier decades: increasing annualised rates of change were less frequent, although rising annualised rates of change still occurred in some locations, particularly for adolescent and younger adult age groups. Rates of stillbirths and under-5 mortality both decreased globally from 1970. Evidence for global convergence of death rates was mixed; although the absolute difference between age-standardised death rates narrowed between countries at the lowest and highest levels of SDI, the ratio of these death rates-a measure of relative inequality-increased slightly. There was a strong shift between 1970 and 2016 toward higher life expectancy, most noticeably at higher levels of SDI. Among countries with populations greater than 1 million in 2016, life expectancy at birth was highest for women in Japan, at 86·9 years (95% UI 86·7-87·2), and for men in Singapore, at 81·3 years (78·8-83·7) in 2016. Male life expectancy was generally lower than female life expectancy between 1970 and 2016, an