Deleterious effect of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino & E.C. Oliveira (Gracilariales, Rhodophyta)

This work evaluated the effects of Tris (hydroxymethyl)-aminomethane (TRIS) buffer and its interaction with nutrient concentration on the development of Gracilaria birdie, a common species on the Brazilian coast that has been exploited for agar production. Responses to different conditions were assessed through growth rates and pigment content (chlorophyll a, phycoerythrin, phycocyanin and allophycocyanin). Provasoli's nutrient solution with and without TRIS addition was tested at concentrations of 12.5, 25 and 50%. The pH was also monitored. G. birdiae grew better in the absence of TRIS and at low nutrient concentrations, 12.5 and 25% (growth rates of 10.8-11.3%.day-1). Higher contents of phycoerythrin and chlorophyll a were observed without TRIS at 12.5 and 25% (Phycoerythrin, 649.6-698.0 μg g-1 fresh biomass; Chlorophyll a, 156.0-168.6 μg g-1 fresh biomass). These findings highlight the deleterious effect of TRIS on growth and phycoerythrin and chlorophyll a content. They also demonstrate the importance of appropriate nutrient concentration for laboratory cultures, depending on the intrinsic characteristics of each species.

Phenology of an agarophyte Gracilaria birdiae Plastino and E.C. Oliveira (Gracilariales, Rhodophyta) in Northeastern Brazil

The reproductive phenology and thallus length of Gracilaria birdiae were studied over a period of 12 months in a natural bed in Northeastern Brazil. Fertile specimens of G. birdiae were observed during the entire study period. Tetrasporophytes were the most common with an annual mean of 80.1±5.6%, followed by cystocarpic plants (9.3±3.4%), male gametophytes (8.3±3.6%) and infertile plants (2.2±3.4%). Only male gametophytes and infertile plants showed a variation in occurrence frequency during the year (p<0.05). With respect to thallus length, a distinct seasonal variation was observed for all reproductive stages (p<0.05), with the highest values recorded during the rainy season (March to August) and the lowest in the dry season (September to February). The results demonstrate that the size of individuals in this population is significantly affected by the periodic changes in the environment caused by rainfall regimes and hydrodynamism.

Histórico de vida de espécimens selvagens e variantes cromáticas de Gracilaria birdiae (Gracilariales, Rhodophyta)

(Gracilariales, Rhodophyta) é uma macroalga marinha de coloração vermelha (vm) comum na costa Nordeste do Brasil e amplamente coletada para a extração de ágar. O objetivo deste estudo foi completar o histórico de vida de espécimens selvagens (coloração vermelha) e variantes cromáticas. Duas variantes foram isoladas, além dos espécimens selvagens coletados na praia de Paracuru, Ceará: i, marrom-esverdeada (me), encontrada em uma população natural da praia de Paracuru; e ii, verde (vd), obtida espontaneamente in vitro a partir de um ramo tetrasporofítico. As condições gerais de cultivo tanto para gametófitos quanto para tetrasporófitos foram: meio de cultura von Stosch 25%; fotoperíodo 14L:10E (luz: escuro); irradiância 100 µm.m-2.s-1 e aeração a cada 30 min. Tetrasporófitos das três colorações e gametófitos verdes foram cultivados também em 10L:14E. O histórico de vida foi completado in vitro em 498 (vm), 523 (me) e 861 (vd) dias, mostrando-se do tipo Polysiphonia. Tetrasporófitos produziram tetrásporos apenas em fotoperíodo 10L:14E. Gametófitos femininos verdes desenvolveram cistocarpos apenas em fotoperíodo 14L:10E. No entanto, a diferenciação de conceptáculos do tipo verrucosa em gametófitos masculinos verdes e a liberação de espermácios ocorreu em ambos os fotoperíodos. Gametófitos masculinos vermelhos apresentaram conceptáculos espermatangiais do tipo henriquesiana, enquanto gametófitos masculinos marrom-esverdeados apresentaram os tipos verrucosa e henriquesiana. Não foram verificadas diferenças quanto ao hábito de espécimens de uma mesma coloração, independentemente do estádio reprodutivo analisado. Verificou-se em todos os tetrasporófitos a germinação de tetrásporos in situ.

Crescimento in vitro de linhagens de coloração vermelha e verde clara de Gracilaria birdiae (Gracilariales, Rhodophyta) em dois meios de cultura: análise de diferentes estádios reprodutivos

Gracilaria sp. é uma das principais macroalgas marinhas atualmente coletadas para produção de ágar no Brasil. Este trabalho objetivou comparar o crescimento in vitro de diferentes estádios reprodutivos tanto de linhagens selvagens (vermelhas), quanto de uma variante cromática (verde clara) de Gracilaria sp., em dois meios de cultura: Von Stosch e Provasoli. Seis linhagens foram selecionadas: gametófitos femininos (F) e masculinos (M), vermelhos (vm) e verde claros (vc); tetrasporófitos vermelhos, porém derivados de cruzamentos distintos, Fvm x Mvm e Fvc x Mvm. As taxas de crescimento avaliadas por 28 dias, foram calculadas a partir de medidas de massa fresca (TCm) e comprimento (TCc). A variante cromática apresentou TCm e TCc inferiores às verificadas para espécimes selvagens. Os dois tipos de meio de cultura resultaram em crescimento satisfatório para as linhagens selvagens. Gametófitos masculinos vermelhos e verde claros e tetrasporófitos vermelhos apresentaram TCm e TCc superiores quando cultivados em Provasoli. Gametófitos femininos verde claros apresentaram TCm semelhantes nos dois meios de cultura, mas as TCs foram superiores em Von Stosch. As TCs de tetrasporófitos derivados de cruzamentos Fvm x Mvm foram superiores às verificadas para tetrasporófitos derivados de cruzamentos Fvc x Mvm.

Deleterious effect of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino & EC Oliveira (Gracilariales, Rhodophyta)

This work evaluated the effects of Tris (hydroxymethyl)-aminomethane (TRIS) buffer and its interaction with nutrient concentration on the development of Gracilaria birdie, a common species on the Brazilian coast that has been exploited for agar production. Responses to different conditions were assessed through growth rates and pigment content (chlorophyll a, phycoerythrin, phycocyanin and allophycocyanin). Provasoli`s nutrient solution with and without TRIS addition was tested at concentrations of 12.5, 25 and 50%. The pH was also monitored. G. birdiae grew better in the absence of TRIS and at low nutrient concentrations, 12.5 and 25% (growth rates of 10.8-11.3%.day(-1)). Higher contents of phycoerythrin and chlorophyll a were observed without TRIS at 12.5 and 25% (Phycoerythrin, 649.6-698.0 mu g g(-1) fresh biomass; Chlorophyll a, 156.0-168.6 mu g g(-1) fresh biomass). These findings highlight the deleterious effect of TRIS on growth and phycoerythrin and chlorophyll a content. They also demonstrate the importance of appropriate nutrient concentration for laboratory cultures, depending on the intrinsic characteristics of each species.

Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta)

Species of Gracilaria are some of the most useful algae in the world for the production of agar. As a consequence of its economic importance, the genus has been the subject of many studies worldwide. Color variants of Gracilaria birdiae have been found in the natural population on the Brazilian coast, and they have also been isolated from plants cultivated in laboratory. These findings raised new questions regarding intraspecific variation and the prospects of cultivating such variants for their agar production. Therefore, this work aimed to determine the mode of color inheritance for two G. birdiae strains: a greenish-brown strain (gb) found in a natural population and a green strain (gr) which had arisen as a spontaneous mutation in a red plant cultured in the laboratory. The pigment contents of these strains, as well as the red wildtype (rd), were also characterized. Crosses between female and male plants of the same color (rd, gr, or gb) and between different colors were performed. Crosses between plants of the same color showed tetrasporophytic and gametophytic descendents of the parental color. Recessive nuclear inheritance was found in the greenish-brown strain, and cytoplasmic maternal inheritance was found in the green strain; both had lower phycoerythrin and higher concentrations of allophycocyanin and phycocyanin than the wild-type. Chlorophyll a contents were similar among all strains. Taken together, our results contribute to knowledge about the variability of this important red algae. In addition, since greenish-brown and green strains showed stability of color, both could be selected and tested in experimental sea cultivation to evaluate if mutants have advantageous performance when compared with red strain.

Influência da profundidade e dos parâmetros ambientais sobre o desenvolvimento de Gracilaria birdiae (Rhodophyta, Gracilariales) em viveiro de camarão

This study evaluates the influence of depth and environmental parameters on the development of Gracilaria birdiae Plastino & Oliveira (Gracilariaceae Rhodophyta) in an organic shrimp pound (Litopenaeus vannamei) under euthrophical conditions. PVC structures (module) witch four ropes laden with 150 g of macroalgae each, were kept during 35 days at three different depths (surface, 10 and 20 cm depth). Wet biomass weighing and environmental parameters (temperature, salinity, turbidity, pH, transparence, precipitation, evaporation, insolation, accumulated solar radiation, nitrite, nitrate, ammonium and orthophosphate) were measured weekly. At all three proposed depths, the macroalgae displayed a higher biomass at the end of experiment than at the initial inoculations. The module kept at a 10 cm depth presented the greatest average biomass (186,3), followed by that kept at 20 cm (180,4 g) and the surface module (169,9 g). Biomass variations showed algae to suffer the direct effects of depths. Biomass loss was associated with the factors that influence light penetration, such as sediment deposits above the thallus, rate of evaporation and precipitation. The smallest loses occurred in the algae kept on surface (0,16%), followed by the algae kept at 20 cm (0,20%) and 10 cm (0,22%). The specific growth rate (SGR) of G. birdiae showed no significant difference between the three depths nor the sample periods. Nevertheless, the modules kept at 10 and 20 cm depths presented similar growth evolution, both growing 0,38%·per day-1, while the module kept on surface had an average SGR of 0,36%·day-1. The models related to growth rate demonstrated temperature, salinity, pH, orthophosphate, ammonium, precipitation and turbidity as the principal environmental parameters influencing the development of G. birdiae

Efeito dos nutrientes (nitrogênio e fósforo) sobre o desenvolvimento da Gracilaria birdiae: crescimento e possível uso como biofiltro

Marine shrimp farming has grown exponentially during the last years in Brazil. In spite of the promising economical situation, this activity is facing an increasing criticism due to its environmental impact. Thus, the necessity of alternatives to mitigate environmental degradation caused by this activity. An alternative that is being studied is the policulture that is the integrated culture of two or more organisms, normally one of them a filtering organism. Among filtering organisms, macroalgae are very practicable because they are efficient in the removal of the exceeding nutrients of the water and do not leave residues in the water. Besides, the integrated culture with macroalgae allows the economical exploration of the seaweed (for the manufacture of jelly and jam, for the dairy industry, pharmaceuticals, etc.) along with possibility of a sustainable aquaculture. In the present experiment, the development of the seaweed Gracilaria birdiae, the influence and tolerance of this species to the environmental parameters, and its absorption efficiency in relation with the three kinds of macronutrients (NH4+, NO3- and PO4-3) found in the effluents of marine shrimp farming was studied. The experiment was divided in two parts: a laboratorial part and one part carried under natural conditions. The water used in the laboratory trial was collected in the shrimp ponds of Tecnarão farm and distributed in aquaria containing 20 g of G. birdiae. In the field trial, 0.5 kg of G. birdiae was inserted in PVC cages cultivated in the farm. The results of the study showed a modest growth of G. birdiae, probably due to its low tolerance to highly eutrophicated environments. However, the removal of nutrients was very expressive. Ammonia was reduced in approximately 34 %. Ortho-phosphate showed a reduction of 93.5 %. The capacity of biofiltration of the NO3- by the macro algae was of 100 %, showing that G. birdiae is a seaweed-filtered with a high level of removal for this nutrient under laboratorial conditions. In spite of the low growth of the macro algae in the experiment, the results in relation to the removal of nutrients of the water was encouraging, suggesting that this species can be an efficient biofilter and thus, a strong candidate to be used in a sustainable aquaculture

Processo de desidratação da alga Gracilaria birdiae beneficiada em laboratório e artesanalmente: análise da atividade antioxidante e composição centesimal

The main aim of this study was to compare the procedure for dehydration of Gracilaria birdiae prepared handmade and laboratory, collected in the northern coast of Rio Grande do Norte. The sample was collected in the Rio do Fogo beach in march 2009. The sample collected followed by two processing, the first the material prepared in laboratory was air-dried at 50°C for 24 hours in air-flow oven. The second the handmade sample was air-dried on the sun during three days. The extract was prepared in three different solvents: ethanol, hydroethanol and water, resulting in ethanol, hidroethanol and aqueous extracts from handmade and laboratory sample. In according with results only the ethanol extract was fractionated yielding the fractions hexane, dichloromethane and ethyl acetate fractions. The different process to obtain Gracilaria birdiae resulted in the samples with different shades. The soluble solids content was higher in the laboratory sample. The chemical composition the both samples were characterized by presenting a considerable amounts of carbohydrates, with amior percentage protein and ash, respectively, in the handmade and laboratory sample. In two samples showed a low content of lipids and the lipid profile showed a higher proportion of monounsaturated fatty acids, with the absence polyunsaturated handmade sample. The phytochemical screening by chemical reactions showed the presence of flavonoids, tannins, alkaloids and saponins the laboratory sample, presenting a greater diversity of bioactive compounds. Through of the analysis by thin layer chromatography was possible to identify the phytosterols β-sitosterol and stigmasterol the both samples, also suggest the presence of β-carotene and chlorophyll α the laboratory sample. The levels of total phenolics and flavonoids were more significant in the ethanol extract of the laboratory sample. The in vitro lethality showed that extracts of the laboratory sample and handmade from 125 to 500 μg/ mL, respectively, were highly lethal. In the evaluation of antioxidant capacity by the system β-carotene/ácido linoleic method and by DPPH radical scavernging assay, the ethanol extract from the laboratory process showed significantly greater activity than the other extracts, being and the first and second methods, respectively, lower and equivalent to the synthetic antioxidant BHT. The handmade ethanol extract has not demonstrated skill in deactivating free radicals, but showed activity in inhibiting lipid peroxidation, although the values were significantly lower than the laboratory sample. We conclude that the dehydration process in the laboratory is the most efficient technique to maintenance of the chemical composition present in the seaweed, providing beneficial properties such as antioxidant capacity. We emphasize that this property can be explored with the objective of adding commercial value to the final product, which will promote the expansion of production of this seaweed in the community of Rio do Fogo

Antioxidant activity and chemical composition of the non polar fraction of Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae (Plastino & Oliveira)

Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae (Plastino & Oliveira) (Gracilariales, Rhodophyta) are seaweeds that occur on the Brazilian coast. Based on their economic and pharmaceutical importance, we investigated the antioxidant activity of the methanolic, ethyl acetate and hexane extracts of both species. The hexane extracts display a high antioxidant activity and comparative analyses indicated G. birdiae as the most active species. Chemical investigation of these fractions showed several carotenoids and fatty acids, as well as cholesterol and sitosterol derivatives. HPLC-DAD analysis of G. birdiae showed violaxanthin (0.04 μg.mg-1 of dry material), antheraxanthin (5.31 μg.mg-1), aloxanthin (0.09 μg.mg-1), zeaxanthin (0.45 μg.mg-1) and β-carotene (0.37 μg.mg-1) as the major carotenoids. G. domingensis showed a similar carotenoid profile, however, with much lower concentration than G. birdiae. Gas chromatography coupled to mass spectrometry was used to determine other nonpolar compounds of these seaweeds. The main compounds detected in both studied species were the fatty acids 16:0; 18:1 Δ9; 20:3 Δ6,9,12, 20:4 Δ5,8,11,14. We found no specificity of compounds in either species. However, G. birdiae, presented higher contents of carotenoids and arachidonic acid than G. domingensis.

Propriedades bioativas dos polissacarídeos sulfatados da alga comestível Gracilaria birdiae

Red marine algae of the genus Gracilaria synthesize sulfated polysaccharides (PS) bioactive. But many of these PS were not properly assessed, as is the case of PS synthesized by edible seaweed Gracilaria birdiae. Previous studies showed that sulfated galactans this alga has anti-inflammatory effect. In this work, a galactan (GB) of G. birdiae was obtained and evaluated by different tests. GB showed anticoagulant activity in APTT assay. GB showed no toxicity to normal cells (3T3), but inhibited the survival of cells of adenocarcinoma of the cervix (HeLa) and human pancreatic cancer (Panc-1) 80% (1.5 mg / ml). GB was not able to hijack the OH radical or the superoxide radical. However, showed activity electron donor in two different tests and presented iron chelator activity (70% and 1.0 mg / ml) and Copper (70% at 0.5 mg / ml). The presence of a higher GB promotes formation of crystals of calcium oxalate dihydrate small size, which is less aggressive, because GB is able to interact with and stabilize the crystal that form. Furthermore, GB (2.0 mg / mL) was not cytotoxic to human renal cells (HEK-293). The data lead us to propose that GB has a great potential for the treatment of urolithiasis

Otimização da extração de polissacarídeos sulfatados da alga vermelha Gracilaria birdiae e análise da atividade anticoagulante e antioxidante

The sulfated polysaccharides (SP) from the edible red seaweed Gracilaria birdiae were obtained using five different condition extraction (GB1: Water; GB1p: Water/proteolysis; GB1s: Water/sonication; GB1sp: Water/sonication/proteolysis; GB2s: NaOH/sonication; GB2sp: NaOH/sonication/proteolysis. The yield (g) increased in the following order GB2sp>GB1sp>GB1p>GB2s>GB1s>GB1. However, the amount of SP extracted increased in different way GB2sp>GB1p>GB1>GB1sp>GB1s>GB2s. Infrared and electrophoresis analysis showed that all conditions extracted the same SP. In addition, monosaccharide composition showed that ultrasound promotes the extraction of other polysaccharides than SP. In the prothrombin time (PT) test, which evaluates the extrinsic coagulation pathway, none of the samples showed anticoagulant activity. While in the activated partial thromboplastin time (aPTT) test, which evaluates the intrinsic coagulation pathway, all samples showed anticoagulant activity, except GB2s. The aPTT activity decreased in the order of GB1sp>GB2sp>GB1p>GB1>GB1s>GB2s. Total capacity antioxidant (TCA) of the SP was also affected by condition extraction, since GB2s and GB1 showed lower activity in comparison to the other conditions. In conclusion, the conditions of SP extraction influence their biological activities and chemical composition. The data showed NaOH/sonication/proteolysis was the best condition to extract anticoagulant and antioxidant SPs from Gracilaria birdiae.

Influência da profundidade e dos parâmetros ambientais sobre o desenvolvimento de Gracilaria birdiae (Rhodophyta, Gracilariales) em viveiro de camarão

This study evaluates the influence of depth and environmental parameters on the development of Gracilaria birdiae Plastino & Oliveira (Gracilariaceae Rhodophyta) in an organic shrimp pound (Litopenaeus vannamei) under euthrophical conditions. PVC structures (module) witch four ropes laden with 150 g of macroalgae each, were kept during 35 days at three different depths (surface, 10 and 20 cm depth). Wet biomass weighing and environmental parameters (temperature, salinity, turbidity, pH, transparence, precipitation, evaporation, insolation, accumulated solar radiation, nitrite, nitrate, ammonium and orthophosphate) were measured weekly. At all three proposed depths, the macroalgae displayed a higher biomass at the end of experiment than at the initial inoculations. The module kept at a 10 cm depth presented the greatest average biomass (186,3), followed by that kept at 20 cm (180,4 g) and the surface module (169,9 g). Biomass variations showed algae to suffer the direct effects of depths. Biomass loss was associated with the factors that influence light penetration, such as sediment deposits above the thallus, rate of evaporation and precipitation. The smallest loses occurred in the algae kept on surface (0,16%), followed by the algae kept at 20 cm (0,20%) and 10 cm (0,22%). The specific growth rate (SGR) of G. birdiae showed no significant difference between the three depths nor the sample periods. Nevertheless, the modules kept at 10 and 20 cm depths presented similar growth evolution, both growing 0,38%·per day-1, while the module kept on surface had an average SGR of 0,36%·day-1. The models related to growth rate demonstrated temperature, salinity, pH, orthophosphate, ammonium, precipitation and turbidity as the principal environmental parameters influencing the development of G. birdiae

Efeito dos nutrientes (nitrogênio e fósforo) sobre o desenvolvimento da Gracilaria birdiae: crescimento e possível uso como biofiltro

Marine shrimp farming has grown exponentially during the last years in Brazil. In spite of the promising economical situation, this activity is facing an increasing criticism due to its environmental impact. Thus, the necessity of alternatives to mitigate environmental degradation caused by this activity. An alternative that is being studied is the policulture that is the integrated culture of two or more organisms, normally one of them a filtering organism. Among filtering organisms, macroalgae are very practicable because they are efficient in the removal of the exceeding nutrients of the water and do not leave residues in the water. Besides, the integrated culture with macroalgae allows the economical exploration of the seaweed (for the manufacture of jelly and jam, for the dairy industry, pharmaceuticals, etc.) along with possibility of a sustainable aquaculture. In the present experiment, the development of the seaweed Gracilaria birdiae, the influence and tolerance of this species to the environmental parameters, and its absorption efficiency in relation with the three kinds of macronutrients (NH4+, NO3- and PO4-3) found in the effluents of marine shrimp farming was studied. The experiment was divided in two parts: a laboratorial part and one part carried under natural conditions. The water used in the laboratory trial was collected in the shrimp ponds of Tecnarão farm and distributed in aquaria containing 20 g of G. birdiae. In the field trial, 0.5 kg of G. birdiae was inserted in PVC cages cultivated in the farm. The results of the study showed a modest growth of G. birdiae, probably due to its low tolerance to highly eutrophicated environments. However, the removal of nutrients was very expressive. Ammonia was reduced in approximately 34 %. Ortho-phosphate showed a reduction of 93.5 %. The capacity of biofiltration of the NO3- by the macro algae was of 100 %, showing that G. birdiae is a seaweed-filtered with a high level of removal for this nutrient under laboratorial conditions. In spite of the low growth of the macro algae in the experiment, the results in relation to the removal of nutrients of the water was encouraging, suggesting that this species can be an efficient biofilter and thus, a strong candidate to be used in a sustainable aquaculture

Influência da profundidade e dos parâmetros ambientais sobre o desenvolvimento de Gracilaria birdiae (Rhodophyta, Gracilariales) em viveiro de camarão

This study evaluates the influence of depth and environmental parameters on the development of Gracilaria birdiae Plastino & Oliveira (Gracilariaceae Rhodophyta) in an organic shrimp pound (Litopenaeus vannamei) under euthrophical conditions. PVC structures (module) witch four ropes laden with 150 g of macroalgae each, were kept during 35 days at three different depths (surface, 10 and 20 cm depth). Wet biomass weighing and environmental parameters (temperature, salinity, turbidity, pH, transparence, precipitation, evaporation, insolation, accumulated solar radiation, nitrite, nitrate, ammonium and orthophosphate) were measured weekly. At all three proposed depths, the macroalgae displayed a higher biomass at the end of experiment than at the initial inoculations. The module kept at a 10 cm depth presented the greatest average biomass (186,3), followed by that kept at 20 cm (180,4 g) and the surface module (169,9 g). Biomass variations showed algae to suffer the direct effects of depths. Biomass loss was associated with the factors that influence light penetration, such as sediment deposits above the thallus, rate of evaporation and precipitation. The smallest loses occurred in the algae kept on surface (0,16%), followed by the algae kept at 20 cm (0,20%) and 10 cm (0,22%). The specific growth rate (SGR) of G. birdiae showed no significant difference between the three depths nor the sample periods. Nevertheless, the modules kept at 10 and 20 cm depths presented similar growth evolution, both growing 0,38%·per day-1, while the module kept on surface had an average SGR of 0,36%·day-1. The models related to growth rate demonstrated temperature, salinity, pH, orthophosphate, ammonium, precipitation and turbidity as the principal environmental parameters influencing the development of G. birdiae