Ecofisiologia de morfotipos reto e espiralado de Cylindrospermopsis raciborskii (Cyanobacteria) em condições controladas

Pós-graduação em Ciências Biológicas (Biologia Vegetal) - IBRC

Culture of the terrestrial cyanobacterium, Nostoc flagelliforme (Cyanophyceae), under aquatic conditions

Both colonies and free-living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free-living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30degreesC at either 20 or 60 mumol photons.m(-2).s(-1); colonies did not develop at 180 mumol photons.m(-2).s(-1), though this light level resulted in the most rapid growth of the cells. Conditions of 60 mumol photons.m(-2).s(-1) and 25degrees C appeared to result in the best colonial development and faster growth of the sheath-held colonies of N. flagelliforme when cultured indoor under aquatic conditions.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

Studies of polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: Structural characterization and effect on the complement system of polysaccharides from Nostoc commune

The cyanobacterium Nostoc commune Vaucher produces quite complex extracellular polysaccharides. The cyanobacterium is nitrogen fixing, and on growing the cyanobacterium in media with and without nitrogen, different types of extracellular polysaccharides were obtained. These were also different from the polysaccharides present in N. commune collected in the field. High pH anion exchange chromatography (HPAEC) of weak acid hydrolysates of the culture-grown material demonstrated that, in this case, HPAEC was useful for comparison of the different polymers. The main differences between the polymers from the field group and the culture-grown samples were the presence of substantial amounts of arabinose, 2-O-methylglucose, and glucuronic acid in the latter. Methylation studies also revealed a difference in the branching points on the glucose units between the field and cultured samples, being 1,4,6 for the first and 1,3,6 for the latter. The field acidic fraction gave, on weak acid hydrolysis and separation on BioGel P2 and HPAEC, 12 oligosaccharide fractions that were isolated and studied by different mass spectroscopy techniques. The structures of the oligosaccharides were determined, and two different series that can originate from two repeating pentamers were identified: GlcAl-4/6GlcM1-4Ga11-4Glc1-4Xyl and GlcAl-4/6Glc1-4Ga11-4Glc1-4Xyl. The difference between these oligosaccharides lies in the methyl substituent on carbon 2 of the glucose unit next to the nonreducing glucuronic acid unit. The polysaccharides from field material were shown to have a strong effect on the complement system.

Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with different colony morphologies: Comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures

Hot water-soluble polysaccharides woe extracted from field colonies and suspension cultures of Nostoc commune Vaucher, Nostoc flagelliforme Berkeley et Curtis, and Nostoc sphaeroides Kutzing. Excreted extracellular polymeric substances (EPS) were isolated from the media in which the suspension cultures were grown. The main monosaccharides of the field colony polysaccharides from the three species were glucose, xylose, and galactose, with an approximate ratio of 2:1:1. Mannose was also present, but the levels varied among the species, and arabinose appeared only in N. flagelliforme. The compositions of the cellular polysaccharides and EPS from suspension cultures were more complicated than those of the field samples and varied among the different species. The polysaccharides from the cultures of N. flagelliforme had a relatively simple composition consisting of mannose, galactose, glucose, and glucuronic acid, but no xylose, as was found in the field colony polysaccharides. The polysaccharides from cultures of N. sphaeroides contained glucose (the major component), rhamnose, fucose, xylose, mannose, and galactose. These same sugars were present in the polysaccharides from cultures of N. commune, with xylose as the major component. Combined nitrogen in the media had no qualitative influence on the compositions of the cellular polysaccharides but affected those of the EPS of N. commune and N. flagelliforme. The EPS of N. sphaeroides had a very low fetal carbohydrate content and thus was not considered to be polysaccharide in nature. The field colony polysaccharides could be separated by anion exchange chromatography into neutral and acidic fractions having similar sugar compositions. Preliminary linkage analysis showed that 1) xylose, glucose, and galactose were 1-->4 linked, 2) mannose, galactose, and xylose occurred as terminal residues, and 3) branch points occurred in glucose as 1-->3,4 and 1-->3,6 linkages and in xylose as a 1-->3,4 linkage. The polymer preparations from field colonies had higher kinematic viscosities than those from corresponding suspension cultures. The high viscosities of the polymers suggested that they might DE suitable for industrial uses.

STUDIES ON MIXED MASS CULTIVATION OF ANABAENA SPP (NITROGEN-FIXING BLUE-GREEN-ALGAE, CYANOBACTERIA) ON A LARGE-SCALE

Studies on mixed mass cultivation of Anabaena spp. on a large scale (5170 m2) were conducted continuously for 3 years. Under the continental monsoon climate in northern subtropics (30-degrees-N, 115-degrees-E), 7-11 g dry weight m-2 day-1 of microalgal biomass on average was harvested in simple plastic greenhouses in the effective growth days during the warmer seasons. The maximum productivity was 22 g m-2 day-1 in the middle of summer. Observations on the productive properties of strains of Anabaena spp. indicated that they were different from and could compensate for each other in their productivities and adaptations to the seasonal changes. With different lining materials (PVC sheets, concrete, sand and soil) in the culture ponds, no significant variation of productivity was found, but bubbling with biogas in the middle of the day and the application of some growth regulating substances (2,4-D, NaHSO3 and extracts of oyster mushroom spawn) was able to improve the production. The cost of microalgal biomass in this way was around 0.75-1.0 US dollar(s) per kilogram.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Effects of light intensity and temperature on Cylindrospermopsis raciborskii (Cyanobacteria) with straight and coiled trichomes: growth rate and morphology

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genetic variability of Brazilian strains of the Microcystis aeruginosa complex (Cyanobacteria/Cyanophyceae) using the phycocyanin intergenic spacer and flanking regions (cpcBA)

The genetic and morphological variability among 15 Brazilian strains of Microcystis aeruginosa (Kütz.) Kütz. collected from four locations was examined and compared with several reference strains of M. aeruginosa, M. viridis (A. Br.) Lemm. and M. wesenbergii (Kom.) Kom. in Kondr. Brazilian strains were classified by morphological features and by comparison of the nucleotide sequences of the cpcBA intergenic spacer and flanking regions. Our results indicate that Brazilian strains classified as M. aeruginosa are phylogenetically diverse compared with reference strains of M. aeruginosa and that the current taxonomy underestimates genetic diversity within M. aeruginosa. The data also demonstrate that morphological criteria alone are inadequate to characterize Microcystis species. Although colonial characters were shown to vary considerably in culture, some genetic lineages demonstrated consistent cellular diameter ranges, indicating that cell size has value as a taxonomic character. The detection of six M. aeruginosa genotypes in a single water body indicates that morphological approaches can also seriously underestimate the diversity of Microcystis bloom populations.

Effects of light intensity and temperature on Cylindrospermopsis raciborskii (Cyanobacteria) with straight and coiled trichomes: growth rate and morphology

Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju (Ordem Nostocales) is one of the most troublesome bloom-forming species in Brazil. Understanding the population dynamics of the different morphotypes of C. raciborskii (straight and coiled) could assist in the prediction of favourable conditions for the proliferation of this potentially toxin-producing species. The aim of the present study was to assess the effects of two different light intensities and temperatures on the growth rate and morphology of the trichomes of the straight and coiled morphotypes. For such, two non-toxin producing strains of C. raciborskii were used - one with a coiled trichome (ITEP31) and another with a straight trichome (ITEP28). The strains were cultured in BG-11 medium in a climatic chamber under controlled conditions. Two light intensities (30 and 90 mu mol.m(-2).s(-1)) were combined at temperatures of 21 and 31 degrees C and the growth rate and morphological changes were analysed. The morphotypes responded differently to the different temperatures and light intensities. Both strains exhibited faster growth velocities when submitted to higher light intensity and temperature. The lower temperature and higher luminosity hampered the development of both strains. Variations in cellular morphology and an absence of akinetes in both strains were related to the lower temperature (21 C). The coiled morphotype demonstrated considerable phenotype plasticity, changing the morphology of trichome throughout its growth curve. Although molecular analysis does not sustain the separation of the morphotypes as distinct species, their different eco-physiological responses should be considered further knowledge of extreme importance for the population control of these potentially toxic organisms.

Laboratory culture studies of Trichodesmium isolated from the great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6 years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR similar to 40-50 mumol quanta m(-2) s(-1)). N-2 fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2-0.3 day(-1) and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR similar to 5-120 mumol quanta m(-2) s(-1)) with the maximum growth occurring at - 40-50 mumol quanta m(-2) s(-1). These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37 psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45 nM. No active growth was observed with the 4.5 nM Fe addition.

Phosphate uptake and growth kinetics of Trichodesmium (Cyanobacteria) isolates from the North Atlantic Ocean and the Great Barrier Reef, Australia

We compared inorganic phosphate (P-i) uptake and growth kinetics of two cultures of the diazotrophic cyanobacterium Trichodesmium isolated from the North Atlantic Ocean (IMS101) and from the Great Barrier Reef, Australia (GBRTRLI101). Phosphate-limited cultures had up to six times higher maximum P-i uptake rates than P-replete cultures in both strains. For strain GBRTRLI101, cell-specific P-i uptake rates were nearly twice as high, due to larger cell size, but P-specific maximum uptake rates were similar for both isolates. Half saturation constants were 0.4 and 0.6 muM for P-i uptake and 0.1 and 0.2 muM for growth in IMS101 and GBRTRLI101, respectively. Phosphate uptake in both strains was correlated to growth rates rather than to light or temperature. The cellular phosphorus quota for both strains increased with increasing P-i up to 1.0 muM. The C:P ratios were 340-390 and N:P ratios were 40-45 for both strains under severely P-limited growth conditions, similar to reported values for natural populations from the tropical Atlantic and Pacific Oceans. The C:P and N:P ratios were near Redfield values in medium with >1.0 muM P-i. The North Atlantic strain IMS101 is better adapted to growing on P-i at low concentrations than is GBRTRLI101 from the more P-i-enriched Great Barrier Reef. However, neither strain can achieve appreciable growth at the very low (nanomolar) P-i concentrations found in most oligotrophic regimes. Phosphate could be an important source of phosphorus for Trichodesmium on the Great Barrier Reef, but populations growing in the oligotrophic open ocean must rely primarily on dissolved organic phosphorus sources.

Antibacterial Activity of Extracellular Products from Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that can be found in freshwater and marine environments as well as in soil. These organisms produce a variety of different biologically active compounds exhibiting anti-bacterial, anti-fungal and anti-cancer properties among others. In this study, cyanobacterial isolates were screened for their ability to produce extracellular antibacterial products. Cyanobacteria were isolated from fresh water and soil samples collected in the Pembroke Pines, FL area. Twenty- seven strains of cyanobacteria were isolated belonging to the following genera: Limnothrix, Nostoc, Fischerella, Anabaena, Pseudoanabaena, Lyngbya, Leptolyngbya, Tychonema, and Calothrix. Individual strains were grown in liquid culture in laboratory conditions. Following 14-day cultivation, the culture liquid was filtered and tested for activity against the following bacteria: Escherichia coli, Bacillus megatarium, Staphylococcus aureus, and Micrococcus luteus. Among all genera of cyanobacterial strains tested, Fischerella exhibited the greatest inhibitory activity. An attempt was made to isolate the active compound from the culture liquid of the active strains. Lipophilic extracts from culture liquid were obtained from three selected Fischerella strains. The extracts proved to have varying levels of activity against the tested bacteria. Inhibitory activity from all three Fischerella strains was detected against B. megatarium and M luteus. The only strain that was active against S. aureus was Fischerella sp. 114-12 while none of the extracts showed activity against E. coli. This kind of screening has potential pharmaceutical and agricultural benefits, including possible discovery of novel antibiotics.

Seawater carbonate chemistry and particulate organic particles during a semicontinuous batch culture experiment with Trichodesmium IMS101, 2007

Diazotrophic (N2-fixing) cyanobacteria provide the biological source of new nitrogen for large parts of the ocean. However, little is known about their sensitivity to global change. Here we show that the single most important nitrogen fixer in today's ocean, Trichodesmium, is strongly affected by changes in CO2 concentrations. Cell division rate doubled with rising CO2 (glacial to projected year 2100 levels) prompting lower carbon, nitrogen and phosphorus cellular contents, and reduced cell dimensions. N2 fixation rates per unit of phosphorus utilization as well as C:P and N:P ratios more than doubled at high CO2, with no change in C:N ratios. This could enhance the productivity of N-limited oligotrophic oceans, drive some of these areas into P limitation, and increase biological carbon sequestration in the ocean. The observed CO2 sensitivity of Trichodesmium could thereby provide a strong negative feedback to atmospheric CO2 increase.

Molecular and biochemical mechanisms involved in the toxicity of a marine cyanobacteria compound on cancer cell lines

In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.