Regional factors as major drivers for microbial community turnover in tropical cascading reservoirs.

The turnover of microbial communities across space is dictated by local and regional factors. Locally, selection shapes community assembly through biological interactions between organisms and the environment, while regional factors influence microbial dispersion patterns. Methods used to disentangle the effects of local and regional factors typically do not aim to identify ecological processes underlying the turnover. In this paper, we identified and quantified these processes for three operational microbial subcommunities (cyanobacteria, particle-attached, and free-living bacteria) from a tropical cascade of freshwater reservoirs with decreasing productivity, over two markedly different dry and rainy seasons. We hypothesized that during the dry season communities would mainly be controlled by selection shaped by the higher environmental heterogeneity that results from low hydrological flow and connectivity between reservoirs. We expected highly similar communities shaped by dispersal and a more homogenized environment during the rainy season, enhanced by increased flow rates. Even if metacommunities were largely controlled by regional events in both periods, the selection had more influence on free-living communities during the dry period, possibly related to elevated dissolved organic carbon concentration, while drift as a purely stochastic factor, had more influence on cyanobacterial communities. Each subcommunity had distinct patterns of turnover along the cascade related to diversity (Cyanobacteria), lifestyle and size (Free-living), and spatial dynamics (particle-attached).

Analysis of major carotenoids and fatty acid composition of freshwater microalgae.

Considering the nutraceutical properties, the high commercial value from pigments and essential lipids and the environmental sustainability, the purposes of this study were to assess the major carotenoids and fatty acids composition of nine microalgae species as a source of nutraceutical compounds and as fatty raw material for biodiesel production. The carotenoid and fatty acid content were analyzed by high performance liquid chromatography tandem mass spectrometry detection method with atmospheric pressure chemical ionization mode (HPLC/APCI-MS/MS) and by high resolution gas chromatography with flame ionization detector (GC-FID). For the carotenoid analysis, the developed method presented a rapid response, a good chromatographic separation, higher sensitivity and can provides more compounds information due the mass spectrum. Among the microalgae evaluated, Desmodesmus protuberans (10.3 mg g-1), Desmodesmus denticulatus var. linearis (8.43 mg g-1) and Chlamydomonas planctogloea (7.4 mg g-1) are good lutein sources. Coelastrum sphaericum (15.29 mg g-1) and Parachlorella kessleri (22.96 mg g-1) showed high astaxanthin content; the others microalgae species presents low carotenoid content. In addition, Chlorella zofingiensis provides high quantities of γ-linolenic acid (4.3%). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels were lower than 1.1 %. Regarding for biodiesel production, the promising strains are Coelastrum sphaericum and Parachlorella kessleri.

Region Growing for Segmenting Green Microalgae Images.

We describe a specialized methodology for segmenting 2D microscopy digital images of freshwater green microalgae. The goal is to obtain representative algae shapes to extract morphological features to be employed in a posterior step of taxonomical classification of the species. The proposed methodology relies on the seeded region growing principle and on a fine-tuned filtering preprocessing stage to smooth the input image. A contrast enhancement process then takes place to highlight algae regions on a binary pre-segmentation image. This binary image is also employed to determine where to place the seed points and to estimate the statistical probability distributions that characterize the target regions, i.e., the algae areas and the background, respectively. These preliminary stages produce the required information to set the homogeneity criterion for region growing. We evaluate the proposed methodology by comparing its resulting segmentations with a set of corresponding ground-truth segmentations (provided by an expert biologist) and also with segmentations obtained with existing strategies. The experimental results show that our solution achieves highly accurate segmentation rates with greater efficiency, as compared with the performance of standard segmentation approaches and with an alternative previous solution, based on level-sets, also specialized to handle this particular problem.

Chemical behavior of Cu, Zn, Cd, and Pb in a eutrophic reservoir: speciation and complexation capacity.

This research aimed at evaluating cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) speciation in water samples as well as determining water quality parameters (alkalinity, chlorophyll a, chloride, conductivity, dissolved organic carbon, dissolved oxygen, inorganic carbon, nitrate, pH, total suspended solids, and water temperature) in a eutrophic reservoir. This was performed through calculation of free metal ions using the chemical equilibrium software MINEQL+ 4.61, determination of labile, dissolved, and total metal concentrations via differential pulse anodic stripping voltammetry, and determination of complexed metal by the difference between the total concentration of dissolved and labile metal. Additionally, ligand complexation capacities (CC), such as the strength of the association of metals-ligands (logK'ML) and ligand concentrations (C L) were calculated via Ruzic's linearization method. Water samples were taken in winter and summer, and the results showed that for total and dissolved metals, Zn > Cu > Pb > Cd concentration. In general, higher concentrations of Cu and Zn remained complexed with the dissolved fraction, while Pb was mostly complexed with particulate materials. Chemical equilibrium modeling (MINEQL+) showed that Zn(2+) and Cd(2+) dominated the labile species, while Cu and Pb were complexed with carbonates. Zinc was a unique metal for which a direct relation between dissolved species with labile and complexed forms was obtained. The CC for ligands indicated a higher C L for Cu, followed by Pb, Zn, and Cd in decreasing amounts. Nevertheless, the strength of the association of all metals and their respective ligands was similar. Factor analysis with principal component analysis as the extraction procedure confirmed seasonal effects on water quality parameters and metal speciation. Total, dissolved, and complexed Cu and total, dissolved, complexed, and labile Pb species were all higher in winter, whereas in summer, Zn was mostly present in the complexed form. A high degree of deterioration of the reservoir was confirmed by the results of this study.

Fourier transform infrared with attenuated total reflectance applied to the discrimination of freshwater planktonic coccoid green microalgae.

Despite the recent advances on fine taxonomic discrimination in microorganisms, namely using molecular biology tools, some groups remain particularly problematic. Fine taxonomy of green algae, a widely distributed group in freshwater ecosystems, remains a challenge, especially for coccoid forms. In this paper, we propose the use of the Fourier Transform Infrared (FTIR) spectroscopy as part of a polyphasic approach to identify and classify coccoid green microalgae (mainly order Sphaeropleales), using triplicated axenic cultures. The attenuated total reflectance (ATR) technique was tested to reproducibility of IR spectra of the biological material, a primary requirement to achieve good discrimination of microalgal strains. Spectral window selection was also tested, in conjunction with the first derivative treatment of spectra, to determine which regions of the spectrum provided better separation and clustering of strains. The non-metric multidimensional scaling (NMDS), analysis of similarities (ANOSIM) and hierarchical clusters (HCA), demonstrated a correct discrimination and classification of closely related strains of chlorophycean coccoid microalgae, with respect to currently accepted classifications. FTIR-ATR was highly reproducible, and provided an excellent discrimination at the strain level. The best separation was achieved by analyzing the spectral windows of 1500-1200 cm(-1) and 900-675 cm(-1), which differs from those used in previously studies for the discrimination of broad algal groups, and excluding spectral regions related to storage compounds, which were found to give poor discrimination. Furthermore, hierarchical cluster analyses have positioned the strains tested into clades correctly, reproducing their taxonomic orders and families. This study demonstrates that FTIR-ATR has great potential to complement classical approaches for fine taxonomy of coccoid green microalgae, though a careful spectrum region selection is needed.

Host-specificity and dynamics in bacterial communities associated with Bloom-forming freshwater phytoplankton.

Many freshwater phytoplankton species have the potential to form transient nuisance blooms that affect water quality and other aquatic biota. Heterotrophic bacteria can influence such blooms via nutrient regeneration but also via antagonism and other biotic interactions. We studied the composition of bacterial communities associated with three bloom-forming freshwater phytoplankton species, the diatom Aulacoseira granulata and the cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii. Experimental cultures incubated with and without lake bacteria were sampled in three different growth phases and bacterial community composition was assessed by 454-Pyrosequencing of 16S rRNA gene amplicons. Betaproteobacteria were dominant in all cultures inoculated with lake bacteria, but decreased during the experiment. In contrast, Alphaproteobacteria, which made up the second most abundant class of bacteria, increased overall during the course of the experiment. Other bacterial classes responded in contrasting ways to the experimental incubations causing significantly different bacterial communities to develop in response to host phytoplankton species, growth phase and between attached and free-living fractions. Differences in bacterial community composition between cyanobacteria and diatom cultures were greater than between the two cyanobacteria. Despite the significance, major differences between phytoplankton cultures were in the proportion of the OTUs rather than in the absence or presence of specific taxa. Different phytoplankton species favoring different bacterial communities may have important consequences for the fate of organic matter in systems where these bloom forming species occur. The dynamics and development of transient blooms may also be affected as bacterial communities seem to influence phytoplankton species growth in contrasting ways.

Cylindrospermopsis raciborskii (Cyanobacteria) exudates: chemical characterization and complexation capacity for Cu, Zn, Cd and Pb.

Cylindrospermopsis raciborskii is a cosmopolitan and potentially toxic planktonic Cyanobacteria that produces and exudes copious amounts of dissolved organic materials. This organism dominates the eutrophic reservoir Barra Bonita (Brazil), where it normally blooms throughout the year. This investigation focused on the characterization of such exudates analyzing their capacity to complex copper, zinc, lead and cadmium through the determination of ligand concentration (CL) and conditional stability constant (logK'ML), as well as elemental composition (C, H, N and S), the content of carbohydrates, proteins, lipids and dissolved organic carbon (DOC). The dissolved organic material was fractionated into 3 molecular weights (>30 kDa; 30-10 kDa; 10-3 kDa) and each fraction was analyzed. The results showed that in the >30 kDa and 30-10 kDa fractions carbohydrates dominate over proteins and lipids. Different CL and logK'ML were obtained for the different molecular weight fractions of the excreted organic materials, suggesting high diversity of ligands. In the >30 kDa, there were more complexing sites (CL) for Cu, but higher affinity (K') for Zn. In the 30-10 kDa fraction, the higher CL was for Cd, but the greatest affinities were for Cu and Zn. In the 10-3 kDa fraction, higher CL was obtained for Cd and Zn, while Cu and Cd had the highest strengths of association. In the environment, such diversity of ligands and strengths of association can result in a displacement of metals weakly bound to the EOM, and increase metal buffering capacity of the environment, supporting higher metal inputs before toxic effects are detected in the biota.

Establishment of a cryopreserved biobank for the Culture Collection of Freshwater Microalgae (CCMA-UFSCar), São Paulo, Brazil / Estabelecimento de um biobanco criopreservado para a Coleção de Culturas de Microalgas de Água-Doce (CCMA-UFSCar), São Paulo, Brasil

Abstract The Culture Collection of Freshwater Microalgae (CCMA-UFSCar, Coleção de Culturas de Microalgas de Água-Doce), based at the Universidade Federal de São Carlos, plays an important role in underpinning Brazilian microalgal research, providing biological materials, substrates and training personnel for a large proportion of the past and current projects in this area. However, recent efforts to expand the culture catalogue/holdings have reached a ceiling because of the logistical practicality of maintaining actively growing cultures. In order to reduce the costs associated with the maintenance regime of cultures, efforts were initiated on the establishment of a cryopreserved biobank for long term maintenance of cultures, thus minimizing the efforts associated with handling of material, as successfully frozen cultures, in theory, could be maintained effectively indefinitely, with the advantage of the stability of cells characteristics. Initial tests were performed on 93 strains including exemplar taxa across the different taxonomic groups in the collection catalogue. The highest levels of success were achieved for the smaller taxa, such as the small green algae; while for the larger and more complex organisms limited to no success was obtained. For the strains tested, over 70% had positive good levels of post-thaw viability and regenerated phenotypically normal cultures.

Resumo A Coleção de Culturas de Microalgas de Água-Doce (CCMA-UFSCar), localizada na Universidade Federal de São Carlos tem um importante papel na sustentação da pesquisa brasileira em microalgas, provendo material biológico, substratos e treinamento de pessoal para uma grande fração dos projetos atuais nessa área. Entretanto, os recentes esforços para expandir o catálogo de culturas atingiram um limite máximo em praticidade logística para a manutenção de culturas metabolicamente ativas. Buscando reduzir os custos do regime de manutenção das culturas, foi-se iniciado o estabelecimento de um banco criopreservado para a manutenção em longo prazo desses organismos, minimizando os gastos com manuseio do material, visto que culturas congeladas com sucesso podem, em teoria, serem mantidas por prazos indeterminados, ainda com a vantagem da estabilidade das características nas culturas. Testes iniciais foram realizados com 93 linhagens, incluindo organismos de diferentes grupos taxonômicos mantidos na coleção de culturas. As maiores taxas de sucesso foram observadas nos organismos menores, como as pequenas algas verdes, enquanto os organismos maiores e mais complexos obtiveram sucesso limitado. Em geral, para as linhagens testadas, acima de 70% dos organismos apresentaram resposta positiva ao processo.

RELEASE OF CARBOHYDRATES AND PROTEINS BY A SUBTROPICAL STRAIN OF RAPHIDIOPSIS BROOKII (CYANOBACTERIA) ABLE TO PRODUCE SAXITOXIN AT THREE NITRATE CONCENTRATIONS(1).

Raphidiopsis brookii P. J. Hill (cyanobacteria) was isolated from a small subtropical eutrophic pond (Biguá Pond) located in the grounds of Rio Grande University in the extreme south of Brazil, following a toxic bloom of this species. Growth, saxitoxin production, and release of carbohydrates and protein were monitored at three sodium nitrate concentrations (500, 1,000, and 1,500 µM), from inoculation up to the stationary growth phase. Growth was monitored by determining the biovolume, chl content, and trichome count. Growth was better described in terms of biovolume and chl measurements, because trichome fragmentation was observed to increase at the stationary growth phase. Carbohydrates and proteins were released in small amounts during most of the experiment, with a significant increase during the stationary phase. Extracellular polysaccharides were essentially composed of glucose, galactose, N-acetyl-glucosamine, mannose, xylose, rhamnose, arabinose, and fucose. The relative proportions of these units showed no significant variation during growth. Small quantities of extracellular free carbohydrates were also detected, and only fucose was released in significant amounts at the lowest nitrate concentration (500 µM). R. brookii produced both saxitoxin and dc-saxitoxin, the former at four times the rate of the latter. This was the first study demonstrating saxitoxin production and the release of both carbohydrate and protein by R. brookii.

Glycolytic activities in size-fractionated water samples: emphasis on rhamnosidase, arabinosidase and fucosidase activities

Glycolytic activities of eight enzymes in size-fractionated water samples from a eutrophic tropical reservoir are presented in this study, including enzymes assayed for the first time in a freshwater environment. Among these enzymes, rhamnosidase, arabinosidase and fucosidase presented high activity in the free-living fraction, while glucosidase, mannosidase and galactosidase exhibited high activity in the attached fraction. The low activity registered for rhamnosidase, arabinosidase and fucosidase in the attached fraction seemed contribute to the integrity of the aggregate and based on this fact, a protective role for these structures was proposed. The presented enzyme profiles and the differences in the relative activities probably reflected the organic matter composition as well as the metabolic requirements of the bacterial community, suggesting that bacteria attached to particulate matter had phenotypic traits distinct from those of free-living bacteria.

Carboidratos dissolvidos do reservatório de Barra Bonita (Estado de São Paulo, Brasil) e sua relação com as algas fitoplanctônicas abundantes / Dissolved carbohydrates from Barra Bonita Reservoir (São Paulo State, Brazil) and its relationships with phytoplanktonic abundant algae

O estudo dos carboidratos dissolvidos e sua relação com as algas abundantes no reservatório de Barra Bonita, foi realizado em coletas mensais (junho de 2002 a janeiro de 2004), em seis profundidades na coluna d'água, cujos valores variaram dependendo da estação do ano e do nível do reservatório, num ponto fixo, localizado 3 km a montante da barragem. Aproximadamente 90 por cento dos carboidratos dissolvidos neste reservatório foram constituídos por carboidratos poliméricos hidrolisáveis (polissacarídeos ou oligossacarídeos). A análise de variância detectou variabilidade entre as concentrações dos carboidratos dissolvidos apenas em escala temporal (p < 0,05), sendo que no eixo vertical, as concentrações destes açúcares apresentaram-se homogêneas (p > 0,05). A composição monossacarídica dos açúcares encontrados no reservatório foi galactose, ramnose, glicose e fucose. Na maioria dos meses amostrados, as concentrações de carboidratos dissolvidos, a maioria deles poliméricos, ocorreram concomitantemente ao registro de florescimentos das espécies abundantes, principalmente cianofíceas, que provavelmente foram as principais fontes de polissacarídeos extracelulares para o sistema.

Dissolved carbohydrates and abundant algae of Barra Bonita Reservoir were studied monthly from June 2002 to January 2004 in six depths whose values vary depending on the seasons and the water level at a sampling site located 3 km upstream the Reservoir dam. Nearly 90 percent of the dissolved carbohydrates were constituted of polymeric hydrolysable carbohydrates (polysaccharides or oligosaccharides). Analysis of variance detected variability among concentrations of dissolved carbohydrates only in temporal scale (p < 0.05), whereas in vertical axis the concentrations of these sugars were uniform (p > 0.05). The monosaccharide composition of the sugars found in the reservoir was galactose, rhamnose, glucose e fucose. During the sampling period, higher concentrations of dissolved carbohydrates, mostly polymeric, occurred together with large phytoplanktonic blooms, predominantly cyanobacteria, which probably were the main source of extracellular polysaccharides in the system.

Some features on the growth of Microcystis aeruginosa Kuetz Emend Elenkin in ASM-1 medium

Alguns aspectos do crescimento de Microcystis aeruginosa foram examinados através da exposiçäo desse organismo a diferentes condiçöes de cultura (luz a 20-C, luz a 40-C, escuro a 20-C e escuro a 4-C) numa tentativa de simular aquelas prováveis situaçöes encontradas no ambiente natural. Os experimentos realizados indicaram que pH, potencial redox, oxigYenio dissolvido e taxa de divisäo celular flutuaram muito além do esperado. Todavia, o glicogênio (energia de reserva) tendeu a desaparecer conteúdo de glicogênio näo diminuiu. A taxa de consumo de glicose foi maior no esturo e apresentou menor valor de Kn, sugerindo maior afinidade da Microcystis para fontes exógenas de glicose. Isto pode ser importante em relaçäo à estratégia de sobrevivência desse organismo sob condiçöes naturais durante o outono, inverno e primavera em lagos temperados

Excreçäo de carboidratos solúveis por microalgas de água doce: I. Comparaçäo de nove diferentes espécies em quatro meios de cultura / Excretion of soluble carbohydrate by freshwater microalgae: I. Comparison of nine different species in four culture media

Neste trabalho é feita a comparaçäo do teor de carboidratos extracelulares em nove espécies de microalgas de água doce: Ankistrodesmus densus, Chlorella homosphaera, Scenedesmus quadricauda, Tetraedron caudatum, Monoraphidium saxatile, Hyalotheca indica, Xanthidium siolii, Chlamydomonas sp e Melosira italica, cultivadas em quatro meios básicos e já clássicos: Chu-10; Bristol; Rhode e WC. Os dados obtidos mostram que as taxas de excreçäo de carboidratos extracelulares säo maiores em meios onde as espécies näo apresentam as melhores taxas de rendimento (células/ml e clorofila-a/1). Isso se deve ou à exaustäo de nutrientes, com algumas espécies, ou ao excesso de nutrientes, com outras

Release of extracellular polysaccaride by spondylosium panduriforme (desmidiaceae)

A desmidiacea filamentosa Spondylosium panduriforme tem uma bainha mucilaginosa relativamente grande e consistente. Usando C14 como marcador foi estudada a liberaçäo de polissacarídeo extracellular solúvel e a marcaçäo dos polissacarídeos da bainha durante 28 horas seguidas. O polissacarídeo solúvel pode ser detectado no meio de cultura uma hora após a adiçäo do marcador enquanto o polissacarídeo da bainha pode ser detectado meia hora depois. Os resultados mostram que a viscosidade no meio de cultura desta espécie näo devida dissoluçäo das bainhas de células velhas, como se pensava. Há evidências que o polissacarídeo solúvel pode ser produzido por um ativo processo fisiológico