Elizabeth Aidar

Distribution of intracellular nitrogen in marine microalgae: Calculation of new nitrogen-to-protein conversion factors

Nitrogen budgets in microalgae are strongly affected by growth conditions and physiological state of the cultures. As a consequence, protein N (PN) to total N (TN) ratio may be variable in microalgae grown in batch cultures, and this may limit the usefulness of the nitrogen-to-protein conversion factors (N-Prot factors), the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of specific N-Prot factors, and experimental data are needed to fill this gap. Complementing a previous study, the present work was designed to quantify the fluctuations of the main nitrogenous compounds during the growth of 12 species of marine microalgae, as well as to determine N-Prot factors for them. The microalgae were cultured in two experimental conditions: (a) using a N-replete culture medium (initial N concentration, 1.18 mM) and aeration, and (b) with a N-depleted culture medium (initial N concentration, 235 μM) and no aeration. The distribution of intracellular nitrogen was studied by constructing budgets of different nitrogen pools in different growth phases of the cultures. In all species, large variations occurred in the distribution of PN and non-protein N (NPN) in the treatments tested and in different growth phases. Intracellular inorganic nitrogen (NO3  − , NO2  −  and NH3 + NH4  + ) was the most important NPN component (0.4 – 30.4% of TN) in all species, followed by nucleic acids (0.3 – 12.2% of TN), and chlorophylls (0.1 – 1.8% of TN). The relative importance of NPN was greater in the exponential phase, decreasing during growth. PN ranged from 59.3 to 96.8% of TN. N-Prot factors are proposed for each of the species studied, based on the ratio of amino acid residues to TN, with values ranging from 2.53 to 5.77. Based on current results and on the previous study, we establish an overall average N-Prot factor for all species, treatments and growth phases of 4.78 ± 0.62 (n = 354). This study confirms that the use of the traditional factor 6.25 is unsuitable for marine microalgae, and the use of the N-Prot factors proposed here is recommended.

DISTRIBUTION OF INTRACELLULAR NITROGEN IN MARINE MICROALGAE : BASIS FOR THE CALCULATION OF SPECIFIC NITROGEN-TO-PROTEIN CONVERSION FACTORS

The utilization of nitrogen‐to‐protein conversion factors (N‐Prot factors) is a widely accepted and practical way to determine total protein content. The accuracy of protein determination depends on the establishment of specific N‐Prot factors, since the conventional factor of 6.25 may be unsuitable for all species. This study was designed to determine the concentrations of the main nitrogenous compounds and to establish N‐Prot factors specific for the following marine microalgae: Chlorella minutissima, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana, Nannochloropsis oculata, Phaeodactylum tricornutum, Prorocentrum minimum, Skeletonema costatum, Synechococcus subsalsus, and Tetraselmis gracilis. Cultures were maintained under a 12‐h photoperiod (300 μmol photons·m−2·s−1) at temperatures of 20.0°± 1.0° C (dark) to 23.0°± 2.0° C (light) in Walne’s culture medium without additional external carbon sources. The distribution of intracellular nitrogen was studied by determining total nitrogen (TN, by CHN [carbon, hydrogen, and nitrogen] analysis), protein N (PN, by analysis of total amino acids), and nonprotein N (NPN, determined by analysis of DNA, RNA, chlorophylls (chl) a,b, and c, and intracellular inorganic nitrogen—NO3−, NO2−, and NH3+ NH4+) in logarithmic and stationary growth phases of cultures. Variations occurred in both accumulation and distribution of PN and NPN among the species, as well as in each species during the different growth phases. Inorganic nitrogen compounds were observed to be the most important NPN source (from 6.4 ± 0.1% to 41.8 ± 4.2% of total N) in all species (except D. tertiolecta), followed by nucleic acids (from 0.8 ± 0.1% to 26.1 ± 2.4% of TN) and chlorophylls (from 0.2 ± 0.0% to 3.1 ± 0.3% of TN). Total amino acid residues ranged from 63.1 ± 4.6% up to 88.1 ± 11.2% of TN, which is in agreement with the presence of high NPN concentrations. N‐Prot factors are proposed for each growth phase in the studied species, based on the ratio of amino acid residues to TN, establishing specific N‐prot factors ranging from 3.60 ± 0.27 to 4.99 ± 0.64. The mean N‐Prot factor for all species/growth phases was 4.58 ± 0.11. The present study shows that the use of the traditional factor 6.25 is not suitable for these marine microalgae, and possibly for other species, because it overestimates their actual protein content.

Effects of different nitrogen sources on the growth and biochemical profile of 10 marine microalgae in batch culture: an evaluation for aquaculture

Abstract Growth responses and biochemical composition were investigated in 10 species of marine microalgae of potential use in aquaculture. The algae were grown in Walnes medium and supplied with nitrate, ammonium or urea as a nitrogen (N) source at a high concentration (1.18 mM). Growth was monitored by daily cell counts, and samples for biochemical analysis were taken near the end of the exponential growth phase, when N nutrient and phosphate still remained in the medium. Two species, Hillea sp. and Prorocentrum minimum, failed to grow with ammonium-N because of the toxic effect of ammonia in high concentration. Hillea sp. and Nannochloropsis oculata on urea-N, and Isochrysis galbana, Phaeodactylum tricornutum and Synechococcus subsalsus on ammonium-N, showed significantly lower final yield than with the other N sources. Greater percentages of protein and chlorophyll (dry matter) occurred in the treatment (N source) that produced higher final cell yield. Treatments producing lower final yields tended to give a larger relative content of carbohydrate and lipid in the cells, and in some cases greater cellular volumes. Seven species tended to produce lower percentages of polyunsaturated fatty acids (PAs) with urea-N. Hillea sp., a microalga not yet used in aquaculture, seems to be a promising food-species because of its fast growth rate with nitrate-N and its large relative content of protein and lipids, and because it produces all essential FAs. Differences in chemical profiles resulted from the use of different nitrogen sources in most cases, but few general trends were identified, even within the same taxonomic group (diatoms, green algae).

Extra and intracelular activities of carbonic anhydrase of the marine microalga Tetraselmis gracilis (Chlorophyta)

As atividades da Anidrase Carbonica (AC) extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinacoes diarias do pH, numero de celulas, atividades enzimaticas, carbono inorgânico total dissolvido (CID) e suas principais especies CO2 e HCO3- foram feitas. A atividade enzimatica aumentou na medida em que a populacao celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentracao de dioxido de carbono decresceu rapidamente, especialmente no terceiro dia do cultivo, quando um significante aumento na atividade enzimatica intracelular foi observado. A concentracao de bicarbonato teve seu maior decrescimo no meio de cultivo no quarto dia, quando a atividade da enzima extracelular teve seu maior aumento, sugerindo seu uso pela alga atraves da atividade da AC. Apos o quarto dia de cultivo, metade das culturas passou a ser aerada com ar atmosferico sem CO2, o que causou um aumento na atividade total e externa da enzima, fazendo com que esses cultivos entrassem na fase estacionaria do crescimento antes que aqueles aerados com ar atmosferico normal. O pH do meio foi medido diariamente, aumentando desde o primeiro ate o quarto dia e permanecendo quase constante ate o fim do cultivo. Material algal transferido para o escuro perdeu toda a atividade enzimatica.

Marine phytoplankton assays: Effects of detergents

Abstract Environmental impacts of an anionic detergent — sodium dodecylbenzene sulfonate (LAS) — a polyphosphate, and a largely employed commercial detergent powder containing methylene blue active substances (MBAS) were evaluated with cultures of the diatom Phaeodactylum tricornutum . Bioassays with natural phytoplankton from an estuarine region (Cananeia — SP) were also performed using the same commercial detergent powder. For P. tricornutum , LC 50 values of LAS were 1.94 and 1.90 mg/l after 48 and 96 h, respectively, and NOEC values were always below 0.50 mg/l. LC 50 values for the commercial detergent were 2.14 mg/l and 1.65 mg/l of its MBAS content after 48 and 96 h, respectively. NOEC values were 1.2 mg MBAS/l after 48 h, and less than 0.1 mg MBAS/l 96 h after the beginning of the experiment. The polyphosphate can be an effective source of phosphorus for cell growth. MBAS concentrations in Cananeia estuarine waters showed values ranging from 0.10 mg/l in surface waters, to 8.75 mg/l in a laundry effluent. The commercial detergent solution added to samples of natural phytoplankton (with or without nutrient enrichment) showed a significant change on chlorophyll-a content, primary production potential, and a drastic reduction in P. tricornutum cell density, at concentrations of 2.0 and 5.0 mg MBAS/l. Microphytoplankton diversity was not affected. The agreement found between field and laboratory experiments emphasized the need for toxicological tests in the monitoring of aquatic environments.

Nitrate reductase activity in the diatom Biddulphia longicruris: characterization and daily oscillation

A atividade de nitrato redutase (NR) foi estudada na diatomacea marinha Biddulphia longicruris. A NR e a enzima responsavel pelo processo de assimilacao de nitrato. O nitrato e reduzido no interior da celula a nitrito pela acao da NR. Esta enzima e normalmente oligomerica e utiliza o NADH como substrato doador de eletrons. Em alguns organismos tambem sao encontradas NRs capazes de utilizarem o NADPH como doador eletronico para a reducao do nitrato a nitrito. Para a diatomacea B. longicmris foram apresentadas evidencias de que a sua NR e especifica para NADH e que a reducao de nitrato em presenca de NADPH nao acontece. Determinacoes in vitro dos valores das constantes de Michaelis-Menten (KM) usando nitrato e NADH como substratos, sao respectivamente 50µ e 80µM. A temperatura otima de reacao enzimatica e a sua dependencia ao pH tambem foram estudadas. Cultivos de B. longicmris foram acompanhados por periodos de 24 horas e foi mostrado que a atividade de NR e encontrada em maiores niveis durante os periodos de transicao de luz/escuro. Os anions nitrito, produtos da reducao de nitrato, sao eliminados pelas celulas nos periodos de maior atividade de NR e se acumulam no meio de cultura. Celulas submetidas a ausencia de nitrato apresentam uma repressao da expressao de NR, sendo ativadas quando pulsos de nitrato sao fornecidos a estas culturas.

Evaluation of produced water toxicity from an oil maritime terminal through Skeletonema costatum toxicity tests

The liquid effluent from an oil maritime terminal, with produced water as the main component, had its toxicity evaluated through toxicity tests with the diatom Skeletonema costatum. Two previously treated effluent samples (effluents A and B), were provided by PETROBRAS for the experiments. Both samples presented high salinity (67‰ for effluent A and 62‰ for effluent B) and low pH values (6.2), whereas total sulphide, phenol and nutrient content, dissolved/dispersed petroleum hydrocarbon concentration, BOD and COD values were quite different from each other. During the toxicity experiment, three replicate flasks with samples for each treatment were exposed to a light radiation of 266µE m2 S-1 and maintained under a 10 h/14 h lightldark cycle, at a temperature of 24 :t 2oC. The EC50 values could not be accurately estimated for effluent A: 60 h and 132 h after starting the experiment they were below 3% and between 3-6% effluent concentration, respectively. Synergistic effects between effluent toxicity and salinity on the growth of S. costatum were detected. The effluent B showed higher toxicity: the EC5O values were 0.17% and 0.40% of effluent concentrations, after 48 h and 96 h, respectively. These results evidenced the deleterious effects of residual organic compounds contained in the aqueous effluents from the oil terminal under the present pretreatment on S. costatum. In the light of the present data, the direct disposal ofthese effluents into Sao Sebastiao Channel waters might be very hazardous to its indigenous biota.

The influence of osmotic shocks on the growth rate and chlorophyll-a content of planktonic algae species

Estudou-se o efeito de variacoes de salinidade sobre as respostas de crescimento e o conteudo de clorofila-α de quatro especies de algas planctonicas (Phaeodactylum tricornutum, Tetraselmis gracilis, Minutocellus polymorphus, Chaetoceros sp), usando a tecnica de culturas estanques. P. tricornutum, apresentando altos valores de taxas maximas de crescimento (div d-1) em todo o gradiente de salinidade experimental, revelou uma alta capacidade de ajuste metabolico quando submetida a choques osmoticos. T. gracilis, M. polymorphus e Chaetoceros sp foram capazes de deslocar o otimo de salinidade para a taxa maxima de crescimento, em funcao da salinidade de pre-cultivo. As quatro especies estudadas mostraram oscilacoes da taxa especifica de crescimento (div d-1) e do conteudo de clorofila-α (pg cel-1), relacionadas aos ciclos de claro-escuro.