Contribuição ao conhecimento sobre a composição de nitrogênio de organismos aquáticos, com ênfase em algas marinhas

Esta pesquisa compreende três estudos independentes envolvendo análises de nitrogênio e substâncias nitrogenadas em organismos aquáticos, com ênfase em algas marinhas. O primeiro estudo consistiu numa comparação dos resultados de análises de nitrogênio total, gerados através do método de Hach, uma alternativa nova e de baixo custo, com análises de composição elementar CHN, um método consagrado, de alto custo. Foram testados 60 organismos aquáticos, compreendendo uma vasta gama de diversidade biológica, como macroalgas, microalgas, crustáceos, peixes, plantas de mangue, plantas de marisma, espermatófitas marinhas, um porífero, um equinodermo e moluscos bivalves, além de duas proteínas puras. Os resultados indicam que as duas técnicas comparadas geram medidas de nitrogênio total, virtualmente idênticas, o que valida plenamente o uso do método de Hach em análises de organismos aquáticos. A distribuição intracelular de nitrogênio e o estabelecimento de fatores de conversão entre nitrogênio e proteínas (fatores N-Prot) para três microalgas marinhas e duas dulciaqüícolas foram os alvos do segundo estudo. As microalgas foram cultivadas em laboratório e constatou-se que as espécies apresentam concentrações variáveis de nitrogênio não-protéico, flutuando entre 5,7% e 17,0% do nitrogênio total. Formas inorgânicas de nitrogênio (nitrato + nitrito + amônia/amônio) foram identificadas como a principal fonte de nitrogênio não-protéico, seguida de ácidos nucléicos e de clorofilas. Concentrações variáveis de ácidos aminados foram encontradas nas espécies testadas. As espécies de microalgas dulciaqüícolas testadas apresentaram concentrações de nitrogênio e de proteínas maiores do que as espécies marinhas, porém as proporções entre nitrogênio não-protéico e nitrogênio total foram semelhantes entre todas as microalgas. Os fatores N-Prot calculados variaram entre 4,70 e 5,48 e foram semelhantes entre microalgas marinhas e dulciaqüicolas pertencentes aos mesmos grupos taxonômicos (clorófitas e cianobactérias). No terceiro estudo, sete macroalgas marinhas foram avaliadas quanto aos teores de nitrogênio e composição de ácidos aminados ao longo de oito coletas sazonais realizadas entre fevereiro de 2005 e janeiro de 2007. As concentrações de nitrogênio total nos talos das sete espécies de macroalgas flutuaram significativamente ao longo do tempo, mas sem padrões cíclicos de variação. Mudanças nas proporções dos ácidos aminados foram virtualmente nulas para cada espécie, mas houve variações significativas nos teores totais de ácidos aminados ao longo do tempo. Fatores de conversão N-Prot foram estabelecidos para cada espécie em cada coleta, variando entre 3,70 e 5,59 e com flutuações insignificantes ao longo do tempo para seis das sete espécies testadas. O uso do tradicional fator N-Prot 6,25 deve ser evitado em algas, sendo recomendados os fatores N-Prot estabelecidos para macroalgas e microalgas nesta pesquisa.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThis research involves three independent studies on nitrogen and nitrogenous substances analyses in aquatic organisms, with emphasis on marine algae. The first study was a comparison of the results for total nitrogen analysis generated by Hach’s method, a new and low-cost alternative, with CHN elemental analysis, a widely accepted and costly method. Sixty aquatic organisms were tested, involving a vast biological diversity, as seaweeds, microalgae, crustaceans, fishes, mangrove plants, salt marsh plants, seagrasses, a marine sponge, an echinoderm and bivalve mollusks, as well as two pure proteins. The results indicate that the two compared techniques yield virtually identical nitrogen measurements, and this validates the Hach’s method for nitrogen analysis of aquatic organisms. The intracellular distribution of nitrogen and the establishment of nitrogen-to-protein conversion factors (N-Prot factors) for three marine and two freshwater microalgae were the targets of the second study. The microalgae were cultured in laboratory and it was noticed that the species showed variable concentrations of non-protein nitrogen, fluctuating from 5.7% to 17.0% of the total nitrogen. Inorganic forms of nitrogen (nitrate + nitrite + ammonia/ammonium) were identified as the main source of non-protein nitrogen, followed by nucleic acids and chlorophylls. Variable concentrations of amino acids were found in the microalgae. The freshwater species showed higher concentrations of both total nitrogen and protein than the marine species, however the proportions between non-protein nitrogen and total nitrogen were similar in all microalgae. The N-Prot factors fluctuated between 4.70 and 5.48 and they were similar with microalgae that belong to the same taxonomic groups (chlorophytes and cyanobacteria). In the third study, seven seaweeds were evaluated regarding total nitrogen and amino acid composition throughout eight seasonal field sampling performed from February 2005 and January 2007. The concentrations of total nitrogen in the thalli of the seven seaweeds fluctuated significantly throughout time, but without a clear cyclic pattern. Changes in the proportions of the amino acid were virtually inexistent for each species, but significant variations in total amino acid were reported throughout time. N-Prot factors were calculated for each species in each sampling, varying from 3.70 and 5.59. No changes in N-Prot factors were found for six out of the seven species throughout time. The use of the traditional N-Prot factor 6.25 should be avoided in algae, and the use of the N-Prot factors established for seaweeds and microalgae in this study is recommended

Crescimento e composição química de dez espécies de microalgas marinhas em cultivos estanques Growth and chemical composition of ten species of marine microalgae in batch cultures

Microalgas apresentam diversas aplicações econômicas consagradas, como usos na aquicultura e na indústria de alimentos, havendo buscas por novos usos, como a geração de biomassa para produção de biodiesel. As possíveis aplicações estão diretamente relacionadas à taxa de crescimento e ao perfil químico das espécies. Assim, a seleção de condições que promovam o aproveitamento da biomassa algácea é fundamental para sua utilização econômica. Neste estudo, 10 espécies de microalgas marinhas foram cultivadas e comparadas quanto ao crescimento e à composição química. Foram observadas diferenças na velocidade de crescimento, com espécies de células menores crescendo mais rapidamente que microalgas maiores. Teores de proteínas, carboidratos, lipídeos e pigmentos fotossintetizantes variaram amplamente entre as espécies, sendo as proteínas as substâncias mais abundantes. Todas as espécies apresentaram concentrações de ácidos aminados semelhantes, sendo os ácidos aspártico e glutâmico os mais abundantes. Algumas espécies apresentaram altas concentrações de ácidos graxos de importância econômica, como os ácidos eicosapentaenoico e linoleico. O balanço dos resultados indica que há poucas tendências gerais relacionadas a grandes grupos taxonômicos.Microalgae show several economic applications, such as uses in aquaculture and in food industry, and there is a search for new uses, such as the biomass production to convert into biodiesel. All possible applications are directly linked to growth rate and the chemical profile of the species. Thus, the selection of conditions to promote a better use of algal biomass is fundamental for economic purposes. In this study, 10 species of marine microalgae were cultured and compared for growth and chemical composition. Remarkable differences of growth performance have been observed, with species with small cell volumes growing faster than species with large cell volumes. Levels of protein, carbohydrate, lipid and photosynthetic pigments varied widely, and proteins were identified as the most abundant substances. Some species showed high concentrations of fatty acids of economic importance, such as eicosapentaenoic and linoleic acids. The concentrations of amino acids were similar among species. In all microalgae, glutamic and aspartic acids were the most abundant amino acids. An overall evaluation of the results indicates that few general trends related to the taxonomy of algal groups were recognized

An assessment of the usefulness of the cyanobacterium Synechococcus subsalsus as a source of biomass for biofuel production

Nowadays algal biofuels are considered one of the most promising solutions of global energy crisis and climate change for the years to come. By manipulation of the culture conditions, many algal species can be induced to accumulate high concentrations of particular biomolecules and can be directed to the desired output for each fuel. In this context, the present study involved the assessment of the effects of CO2 availability and nitrogen starvation on growth and chemical composition of the cyanobacterium Synechococcus subsalsus, testing a fast-growing native strain. The control experiments were performed with Conway culture medium in 12-day batch cultures, in 6-liter flasks and 12 h photoperiod, with addition of 2 L min-1 filtered air to each flask. Other two experimental conditions were also tested: (i) the placement into the cultures of additional dissolved nutrients except nitrogen, one week after the start of growth (N-), and (ii) the input of pure CO2 into the flasks from the 5th day of growth (C+). In all cultures, daily cell counts were done throughout the cultivation, as well as measurements of pH and cell biovolumes. Maximum cell yield were found in N-experiments, while cell yields of C+ and control were similar. Dissolved nitrogen was exhausted before the end of the experiments, but dissolved phosphorus was not totally consumed. Protein and chlorophyll-a concentrations decreased from the exponential to the stationary growth phase of all experiments, except for protein in the control. In all experiments, carbohydrate, lipid and total carotenoid increased from the exponential to the stationary growth phase, as an effect of nitrogen limitation. Increments in carbohydrate concentrations were remarkable, achieving more than 42% of the dry weight (dw), but concentrations of lipid were always lower than 13% dw. The addition of pure CO2 did not cause a significant increase in biomass of S. subsalsus nor generated more lipid and carbohydrate than the other treatments. Nitrogen starvation caused an intense accumulation of carbohydrate, but the increments of lipid were small. Despite the fast growth, the cyanobacterium S. subsalsus has a virtually null potential for biodiesel production, given its low lipid concentrations. The high concentrations of carbohydrate combined with fast growth point to the potential use of this species as raw material for other possible biotechnological processes, after a demonstration of technical and economic viability

Proximate composition of marine invertebrates from tropical coastal waters, with emphasis on the relationship between nitrogen and protein contents

The chemical profiles of Desmapsamma anchorata, Hymeniacidon heliophila (Porifera), Bunodosoma caissarum, Renilla muelleri (Cnidaria), Aplysia brasiliana, Eledone massyae, Isognomon bicolor (Mollusca), Echinaster brasiliensis, Echinometra lucunter, Holothuria grisea, Lytechinus variegatus (Echinodermata), and Phallusia nigra (Chordata) were determined. Hydrosoluble protein was the most abundant class of substances for all species, except for the ascidian Phallusia nigra, in which the carbohydrate content was higher. The percentages of hydrosoluble protein (dry weight, dw) varied widely among the invertebrates, ranging from 5.88% (R. muelleri) to 47.6% (Eledone massyae) of the dw .The carbohydrate content fluctuated from 1.3% (R. muelleri) to 18.4% (Aplysia brasiliana) of the dw. For most of the species, lipid was the second most abundant class of substances, varying from 2.8% (R. muelleri) to 25.3% (Echinaster brasiliensis) of the dw. Wide variations were also found for the invertebrates nitrogen content, with the lowest value recorded in the cnidarian R. muelleri (2.02% of the dw) and the highest in the molluscan E. massyae (12.7% of the dw). The phosphorus content of the dw varyed from 0.24% (R. muelleri) to 1.16% (E. massyae). The amino acid composition varied largely among the species, but for most of the species glycine, arginine, glutamic acid, and aspartic acid were the most abundant amino acids, with histidine and tyrosine among the less abundant amino acids. The actual content of total protein in the samples was calculated by the sum of amino acid residues, establishing dw values that fluctuated from 11.1% (R. muelleri) to 66.7% (E. massyae). The proteinaceous nitrogen content was high in all species, with an average value of 97.3% of the total nitrogen. From data of total amino acid residues and total nitrogen, specific nitrogen-to-protein conversion factors were calculated for each species. The nitrogen-to-protein conversion factors ranged from 5.10 to 6.15, with an overall average of 5.45. The use of the specific nitrogen-to-protein conversion factors established here is recommended, since it would yield more accurate determinations of total protein in the species tested in this study

Influence of laboratory conditions on two species Dictyotaceae family (Phaeophyceae): Diterpenoid profile and physiological response

The seaweed Dictyotaceae family has a great wealth of diterpenes with important biological activities. Information on these diterpenes qualitative and quantitative aspects in conjunction with physiological data and abiotic parameters are essential to obtain an effective algal culture system that is a sustainable resource. In this study, the diterpene profile of Dictyota menstrualis and Canistrocarpus cervicornis were analyzed as a function of in vitro maintenance time and the influence of culture medium. The algae were collected in the state of Rio de Janeiro and subjected to an acclimation period in the laboratory. Then, the species were acclimated in aquaria containing seawater from the collection sites. Then biomass algal was divided into (i) control experiment (CC): Growth in sterilized seawater; (ii) enriched experiment (EE): Growth in sterilized seawater enriched with Provasoli/2. Triplicate experiments were maintained in vitro (2.4% m/v), and the medium was changed every 7 days. Diterpenes were monitored using dichloromethane extracts from dried algae: (a) collected in the field, (b) after the acclimation period, and (c) from experimental units taken every 7 days for a total of 21 days. Among the main results, we found response variables such as specific growth rate (SGR); moreover, positive data were obtained for the alga C. cervicornis in CC, and this was evident showing after 7 days of cultivation in the experimental control unit (CU). For D. menstrualis, the SGRs for the treatments were negative at the monitored time, which was due to biomass losses, frond breakage, and rapid decomposition of some individuals that did not withstand the stress conditions during their collection. The analysis of the chemical profile by GC-MS made it possible to monitor 12 diterpenes and one sterol in the alga D. menstrualis and 7 diterpenes in C. cervicorni. The correlations found by Generalized Linear Models (GLMs) and redundancy analysis (RDA) statistical analyzes between the diterpene profile with the monitored variables (Nitrate, ammonia and pH), showed correlations for 10 diterpenes of the species D. menstrualis and 1 of the C. cervicornis, our analyses are important for the discussion of metabolic plasticity of the Dictyotaceae famil