Uso potencial de la macroalga verde Ulva clathrata en el cultivo de camarón blanco litopenaeus vannamei.

Tesis (Doctor en Ciencias con Especialidad en Biotecnología) UANL, 2011.

Rapid and slow modulation of nitrate reductase activity in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.

Dislodgment and attachment strength of the intertidal macroalga Hormosira banksii (Fucales, Phaeophyceae)

Hormosira banksii is a dominant intertidal alga characterised by limited dispersal of propagules, yet it has a broad distribution throughout temperate Australasia. However, the high abundance of beach wrack of H. banksii on sandy beaches along southwest Victoria, the frequency of frond dislodgment and the force required to break it from the substratum remain unknown. Dislodgment of macroalgae has been shown to influence dispersal processes; therefore, we tested the model that long-distance dispersal of H. banksii is facilitated by weak attachment to the substratum and frequent dislodgment of fertile fronds. We monitored the dislodgment of H. banksii individuals and conducted in situ pull tests to determine the attachment strength of fronds of H. banksii. We further tested whether thallus size or pull direction influenced attachment strength of H. banksii. We found that breakage of vesicles and fronds was a regular event during the survey period and that the mean attachment strength of fronds of H. banksii was weak compared to other intertidal algae. Furthermore, we found that thallus size did not influence the force required to break fronds of H.banksii from the substratum but that the direction that fronds were pulled from the substratum did. We suggest that fronds of H. banksii do drift frequently and that long-distance dispersal is likely to be an important mechanism for the distributional success of this species.

Utilização da macroalga Ulva lactuca linnaeus na redução de nutrientes (NH4+,NO3- e PO4-2) provenientes da carcinicultura

Dentre as macroalgas capazes de absorver altas concentrações de N e P dissolvidos na água, destaca-se a Chlorophyta Ulva lactuca, bastante adaptável e resistente às adversidades ambientais, como grandes variações de temperatura, salinidade, matéria orgânica e metais pesados. Trata-se também de uma espécie bastante comum nas áreas intertidais do litoral norte-riograndense. Devido a suas características ecológicas, fisiológicas e nutricionais, foi avaliado nesse estudo, o seu potencial como biofiltro na redução de NH4+, NO3- e PO4-2, tanto em condições controladas como também em um viveiro de camarão. No experimento laboratorial, foram utilizados quatro aquários de vidro de 30 x 20 x 20cm com 10L de água, sendo três aquários experimentais contendo 20g de U. lactuca e um controle. O acréscimo de biomassa foi de 2,92g (22,92 ± 6,29g; p < 0,05) em relação ao inóculo inicial de 20g, sob temperatura (28,50 ± 0,58ºC), salinidade (35,00 ± 0,00 ), pH (8,26 ± 0,02) e luz constante (250 μmol.m2s-1). O crescimento positivo (1,78 ± 4,38%dia-1; p < 0,05), juntamente com a alta eficiência de absorção de amônio (83%; p < 0,001), nitrato (83%; p < 0,001) e ortofosfato (53%; p < 0,001), demonstrou que, nessas condições, a Ulva lactuca absorveu os nutrientes e aumentou sua biomassa. Já no experimento de campo, realizado na fazenda TECNARÃO, situada no município de Arez/RN (06° 11 40 Latitude Sul, e 35º 09 37 Longitude Oeste), foram utilizadas três gaiolas de PVC, posicionadas a 12cm da superfície da água, cada uma com dimensões de aproximadamente 59 x 59 x 15cm, onde foram colocadas 200g de U. lactuca. O ganho de biomassa de 3g (203,00 ± 41,02g; p < 0,001) foi muito semelhante às condições controladas, demonstrando a adaptabilidade da espécie em condições ambientais variáveis, onde, apesar da temperatura pouco variável (27,45 ± 0,64ºC), houve progressiva diminuição de salinidade (25 - 15 ), devido ao período de fortes chuvas (34,70 ± 23,78mm). Somado a isso, foram observados vários fatores biológicos interferindo no viveiro, como a presença de epífitas, organismos endofíticos, fouling e a herbivoria por parte dos próprios camarões. Houve aumento nas concentrações de NH4+ (4,36 ± 1,69 μmol.L-1), NO3- (0,17 ± 0,25μmol.L-1) e PO4-2 (0,41 ± 0,13μmol.L-1), coincidindo com o crescimento da espécie até a terceira semana. Todos os parâmetros ambientais analisados, assim como a biomassa e a Taxa de Crescimento Relativo (TCR), obtidos no campo, apresentaram variações altamente significativas (p < 0,001). As correlações observadas entre biomasa e NH4+ (r = 0,82; p < 0,001) e entre biomassa e PO4-2 (r = 0,87; p < 0,001), indicam que esta espécie é capaz de ter um crescimento satisfatório nas condições eutróficas de um viveiro de camarão, sendo possível seu uso como biofiltro.

Estudo do crescimento, eficiência de biofiltração e cinética de absorção de nutrientes (N-NH, N-NO e P-PO4³) da macroalga Gracilaria cervicornis (Turner) J. Agardh

The objective of this study was to examine the growth of Gracilaria cervicornis cultured in a shrimp (Litopenaeus vannamei) pond and to determine the absorption efficiency and the kinetics parameters (Vmax, Ks e Vmax:Ks) of this macroalgae for the nutrients N-NO3-, N-NH4+ and P-PO4-3, aiming at its use as bioremediatory of eutrophicated environments. For this study, two experiments (field and laboratory) were developed. In the field study, the seaweed was examined in relation to the growth and the biomass. In the laboratory experiment, the absorption efficiency of G. cervicornis was measured through the monitoring of the concentration of the three nutrients (N-NO3-, N-NH4+ e P-PO4-3) during 5 hours and the kinetic parameters were determined through the formula of Michaelis-Menten. The results obtained in this study demonstrated that G. cervicornis benefited from the available nutrients in the pond, increasing 52.4% of its biomass value after 30 days of culture. It was evidenced that the variability of the biomass could be explained through the salinity, availability of light (transparency and solid particle in suspension) and concentration of N-NO3- in the environment. In the laboratory experiment, the highest absorption efficiency was found in the treatments with low concentration (5 µmol.L-1), being evidenced a reduction of up to 85,3%, 97,5% and 81,2% of N-NH4+, N-NO3- and P-PO43-, respectively. Regarding the kinetic parameters, G. cervicornis presented better ability in absorbing N-NH4+ in high concentrations (Vmax = 158,5 µmol g-1 dry wt h-1) and P-PO43- in low concentrations (Ks = 5 µmol.L-1 e Vmax:Ks = 10,3). The results of this study show that G. cervicornis could be cultivated in shrimp ponds, presents a good capacity of absorption for the tested nutrients and is a promising candidate for biorremediation in shrimp pond effluent

Influence of temperature, light and nutrients on the growth rates of the macroalga Gracilaria domingensis in synthetic seawater using experimental design

In the present study, the daily relative growth rates (DRGR, in percent per day) of the red macroalga Gracilaria domingensis in synthetic seawater was investigated for the combined influence of five factors, i.e., light (L), temperature (T), nitrate (N), phosphate (P), and molybdate (M), using a statistical design method. The ranges of the experimental cultivation conditions were T, 18-26A degrees C; L, 74-162 mu mol photons m(-2) s(-1); N, 40-80 mu mol L-1; P, 8-16 mu mol L-1; and M, 1-5 nmol L-1. The optimal conditions, which resulted in a maximum growth rate of a parts per thousand yen6.4% d(-1) from 7 to 10 days of cultivation, were determined by analysis of variance (ANOVA) multivariate factorial analysis (with a 2(5) full factorial design) to be L, 74 mu mol photons m(-2) s(-1); T, 26A degrees C; N, 80 mu mol L-1; P, 8 mu mol L-1; and M, 1 nmol L-1. In additional, these growth rate values are close to the growth rate values in natural medium (von Stosch medium), i.e., 6.5-7.0% d(-1). The results analyzed by the ANOVA indicate that the factors N and T are highly significant linear terms, X (L), (alpha = 0.05). On the other hand, the only significant quadratic term (X (Q)) was that for L. Statistically significant interactions between two different factors were found between T vs. L and N vs. T. Finally, a two-way (linear/quadratic interaction) model provided a quite reasonable correlation between the experimental and predicted DRGR values (R (adjusted) (2) = 0.9540).

Effects of increased CO2 levels on growth, photosynthesis, ammonium uptake and cell composition in the macroalga Hypnea spinella (Gigartinales, Rhodophyta)

[EN] The red seaweed Hypnea spinella (Gigartinales, Rhodophyta), was cultured at laboratory scale under three different CO2 conditions, non-enriched air (360 ppm CO2)and CO2-enriched air at two final concentrations (750 and 1,600 ppm CO2), in order to evaluate the influence of increased CO2 concentrations on growth, photosynthetic capacity, nitrogen removal efficiency, and chemical cellular composition. Average specific growth rates of H. spinella treated with 750 and 1,600 ppm CO2-enriched air increased by 85.6% and 63.2% compared with non-enriched air cultures. CO2 reduction percentages close to 12% were measured at 750 ppm CO2 with respect to 5% and 7% for cultures treated with air and 1,600 ppm CO2, respectively. Maximum photosynthetic rates were enhanced significantly for high CO2 treatments, showing Pmax values 1.5-fold higher than that for air-treated cultures. N–NH4+ consumption rates were also faster for algae growing at 750 and 1,600 ppm CO2 than that for non-enriched air cultures. As a consequence of these experimental conditions, soluble carbohydrates increased and soluble protein contents decreased in algae treated with CO2-enriched air. However, internal C and N contents remained constant at the different CO2 concentrations. No significant differences in data obtained with both elevated CO2 treatments, under the assayed conditions, indicate that H. spinella is saturated at dissolved inorganic carbon concentrations close by twice the actual atmospheric levels. The results show that increased CO2 concentrations might be considered a key factor in order to improve intensively cultured H. spinella production yields and carbon and nitrogen bioremediation efficiencies.

Molecular techniques for gene characterization: iPCR for identification of the 5'UTR of the ODC gene in the red macroalga Grateloupia imbricata

Máster Oficial en Gestión Costera

Respiratory metabolism in the marine macroalga, Ulva spp: exploratory studies with the respiratory electron transport system (ETS)

Máster en Oceanografía

Diurnal fluctuations in seawater pH influence the response of a calcifying macroalga to ocean acidification

Coastal ecosystems that are characterized by kelp forests encounter daily pH fluctuations, driven by photosynthesis and respiration, which are larger than pH changes owing to ocean acidification (OA) projected for surface ocean waters by 2100. We investigated whether mimicry of biologically mediated diurnal shifts in pH-based for the first time on pH time-series measurements within a kelp forest-would offset or amplify the negative effects of OA on calcifiers. In a 40-day laboratory experiment, the calcifying coralline macroalga, Arthrocardia corymbosa, was exposed to two mean pH treatments (8.05 or 7.65). For each mean, two experimental pH manipulations were applied. In one treatment, pH was held constant. In the second treatment, pH was manipulated around the mean (as a step-function), 0.4 pH units higher during daylight and 0.4 units lower during darkness to approximate diurnal fluctuations in a kelp forest. In all cases, growth rates were lower at a reduced mean pH, and fluctuations in pH acted additively to further reduce growth. Photosynthesis, recruitment and elemental composition did not change with pH, but ?(13)C increased at lower mean pH. Including environmental heterogeneity in experimental design will assist with a more accurate assessment of the responses of calcifiers to OA.

Diffusion boundary layers ameliorate the negative effects of ocean acidification on the temperate coralline macroalga Arthrocardia corymbosa

Anthropogenically-modulated reductions in pH, termed ocean acidification, could pose a major threat to the physiological performance, stocks, and biodiversity of calcifiers and may devalue their ecosystem services. Recent debate has focussed on the need to develop approaches to arrest the potential negative impacts of ocean acidification on ecosystems dominated by calcareous organisms. In this study, we demonstrate the role of a discrete (i.e. diffusion) boundary layer (DBL), formed at the surface of some calcifying species under slow flows, in buffering them from the corrosive effects of low pH seawater. The coralline macroalga Arthrocardia corymbosa was grown in a multifactorial experiment with two mean pH levels (8.05 'ambient' and 7.65 a worst case 'ocean acidification' scenario projected for 2100), each with two levels of seawater flow (fast and slow, i.e. DBL thin or thick). Coralline algae grown under slow flows with thick DBLs (i.e., unstirred with regular replenishment of seawater to their surface) maintained net growth and calcification at pH 7.65 whereas those in higher flows with thin DBLs had net dissolution. Growth under ambient seawater pH (8.05) was not significantly different in thin and thick DBL treatments. No other measured diagnostic (recruit sizes and numbers, photosynthetic metrics, %C, %N, %MgCO3) responded to the effects of reduced seawater pH. Thus, flow conditions that promote the formation of thick DBLs, may enhance the subsistence of calcifiers by creating localised hydrodynamic conditions where metabolic activity ameliorates the negative impacts of ocean acidification.

Seawater carbonate chemistry and growth, reproduction of calcifying and non-calcifying epibionts of a brown macroalga in a laboratory experiment

Seaweeds are key species of the Baltic Sea benthic ecosystems. They are the substratum of numerous fouling epibionts like bryozoans and tubeworms. Several of these epibionts bear calcified structures and could be impacted by the high pCO2 events of the late summer upwellings in the Baltic nearshores. Those events are expected to increase in strength and duration with global change and ocean acidification. If calcifying epibionts are impacted by transient acidification as driven by upwelling events, their increasing prevalence could cause a shift of the fouling communities toward fleshy species. The aim of the present study was to test the sensitivity of selected seaweed macrofoulers to transient elevation of pCO2 in their natural microenvironment, i.e. the boundary layer covering the thallus surface of brown seaweeds. Fragments of the macroalga Fucus serratus bearing an epibiotic community composed of the calcifiers Spirorbis spirorbis (Annelida) and Electra pilosa (Bryozoa) and the non-calcifier Alcyonidium hirsutum (Bryozoa) were maintained for 30 days under three pCO2 conditions: natural 460±59 µatm, present-day upwelling1193±166 µatm and future upwelling 3150±446 µatm. Only the highest pCO2 caused a significant reduction of growth rates and settlement of S. spirorbis individuals. Additionally, S. spirorbis settled juveniles exhibited enhanced calcification of 40% during daylight hours compared to dark hours, possibly reflecting a day-night alternation of an acidification-modulating effect by algal photosynthesis as opposed to an acidification-enhancing effect of algal respiration. E. pilosa colonies showed significantly increased growth rates at intermediate pCO2 (1193 µatm) but no response to higher pCO2. No effect of acidification on A. hirsutum colonies growth rates was observed. The results suggest a remarkable resistance of the algal macro-epibionts to levels of acidification occurring at present day upwellings in the Baltic. Only extreme future upwelling conditions impacted the tubeworm S. spirorbis, but not the bryozoans.

Extração, caracterização e prospecção de atividades farmacológicas de polissacarídeos sulfatados da macroalga verde Caulerpa prolifera

This study aimed to extract, characterize and conduct a prospective analysis of pharmacological activities of sulfated polysaccharides from green seaweed Caulerpa prolifera. Seven fractions (CP-0.3/CP-0.5/CP-0.7/CP-0.9/CP-1.1/CP-1.5/CP-2.0) were obtained from C. prolifera by alkaline proteolysis followed by sequential precipitation in acetone. The physicochemical analyzes indicated that C. prolifera synthesizes a homogalactan (CP-0.9) and different populations of sulfated heteropolysaccharides. In the analysis of anticoagulant activity, all fractions except CP-0.3, influenced the intrinsic coagulation pathway. All fractions showed antioxidant activity in six different assays being more pronounced in hydrogen peroxide scavenging assay, especially CP-0.3, CP-0.7 and CP-0.9 (which obtained 61% of hydrogen peroxide scavenging), in ferric chelation assay (especially CP-0.9 with 56% chelation) and cupric chelation assay (especially CP-2.0 with 78% chelation). With respect to immunomodulatory activity, the presence of CP-0.3, CP-0.7 and CP-0.9 showed an immunogenic potential, increasing the production of nitric oxide (NO) by 48, 142 and 163 times, respectively. Conversely, the NO synthesis fell 73% after the activation of macrophages by LPS, incubated concurrently with CP-2.0. The anti-adipogenic activity of the fractions was also evaluated and CP-1.5 was able to reduce the differentiation of pre-adipocytes (3T3-L1) into adipocytes by 60%, without affecting the cell viability. The fractions CP-0.3, CP-0.5 and CP-0.9 reduced the viability of the HeLa cells (human cervical adenocarcinoma) by 55% and CP-1.5 reduced the viability of the 786-0 cells (human renal adenocarcinoma) by 75%. Leishmanicidal activity and microbicide effect against Carbapenem-resistant Klebsiella pneumoniae (KPC) have not been identified. However, the viability of Staphylococcus epidermidis was reduced by 23.8% in the presence of CP -1.5. All fractions were able to change the formation of calcium oxalate crystals. CP-0.3, CP-0.5 and CP-1.1 only promoted the formation of COD type crystals with a very small size (1 μm). Confocal microscopy and zeta potential data of crystals formed in the presence of the samples showed that the polysaccharides present in the fractions must interact with calcium ions present throughout the crystal lattice, affecting the growth and morphology of crystals The results described herein indicate that the fractions rich in polysaccharides obtained from the green seaweed C. prolifera present a multi therapeutic potential, and subsequent purification steps, as well as research on the mechanisms of action by which these polymers act should be investigated.