Armando Augusto Henriques Vieira

MUCILAGINOUS CAPSULE ADSORPTION AND INTRACELLULAR UPTAKE OF COPPER BY KIRCHNERIELLA APERTA (CHLOROCOCCALES)1

The purpose of the present investigation was to evaluate possible ecological and physiological functions of mucilaginous capsules produced by the freshwater algae Kirchneriella aperta Teiling (Chlorococcales) as related to copper ions. All experiments were performed using synthetic media under laboratory‐controlled conditions. Copper interactions were investigated by distinguishing between adsorption onto the mucilaginous material present at the surface of the cells, intracellular uptake, and differentiation between total dissolved copper and free copper ions in the culture medium. Kirchneriella aperta is sensitive to copper, as revealed by a 96‐h EC50 value of 10−9.22 M Cu2+. We demonstrated that the mucilaginous capsules were able to sequester copper ions from the medium through a passive mechanism, thus providing the cell with a mechanism able to postpone the toxic effects of copper. The organic material that diffuses into the test medium as well as the mucilaginous capsules produced by K. aperta both effectively complex copper; thus, toxicity must be related to free copper ions and not the total dissolved copper concentration in the medium.

RELATIVE INCREASE OF DEOXY SUGARS DURING MICROBIAL DEGRADATION OF AN EXTRACELLULAR POLYSACCHARIDE RELEASED BY A TROPICAL FRESHWATER THALASSIOSIRA SP. (BACILLARIOPHYCEAE)1

The aim of this study was to characterize the extracellular polysaccharides (EPS) released by a freshwater Thalassiosira sp. (Bacillariophyceae) and evaluate their degradation by heterotrophic microbial populations from the same habitat of Thalassiosira sp., a tropical eutrophic reservoir. The EPS were purified by anion exchange column chromatography, the monosaccharide composition was determined by GC, and the linkages of the monosaccharides by GC‐MS. The EPS is a mannose‐rich heteropolysaccharide composed of two different acidic fractions. Both of these fractions are composed of mannose, rhamnose, fucose, xylose, galactose, glucose, glucuronic acid, and N‐acetyl glucosamine but with different proportions. N‐acetyl galactosamine occurs only in fraction 1 and galacturonic acid only in fraction 2. We monitored the concentrations of the monosaccharides in the EPS during its degradation using pulse amperometric detection in an HPLC. The decay patterns of the monosaccharides were varied and the deoxy sugars, fucose and rhamnose, were degraded at a slower rate than the other components, increasing their relative concentrations and the hydrophobic feature of the EPS. The possibility of a selective degradation, which enhances the stickiness of the EPS, promoting transparent exopolymeric particles and aggregate formation, is discussed based on the literature data.

A rapid spectrophotometric method for the determination of transparent exopolymer particles (TEP) in freshwater.

A simple and rapid spectrophotometric method is proposed for the determination of transparent exopolymer particles (TEP) in freshwater samples. In this method, TEP reacts with excess of alcian blue solution yielding a low solubility dye-TEP complex. After centrifugation, the concentration of the remaining dye in the supernatant was determined at 602nm and its concentration was related to the concentration of TEP in freshwater. The effect of alcian blue concentration from 1.5x10(-3) to 9.0x10(-3)% (m/v), solution pH from 2.5 to 6.9 and stirring time from 20 to 120s on the analytical curve was investigated. Under the optimum conditions established, such as alcian blue concentration of 3.0x10(-3)% (m/v); pH of 4.0 (0.2moll(-1) acetate buffer solution) and stirring time of 1min, the analytical curve was linear from 0.50 to 10mugml(-1) (A=0.34-0.037[GX]; r(2)=0.9999; where A is the absorbance and [GX] the gum xanthan concentration in mugml(-1)) with a detection limit of 0.10mugml(-1). The recovery of TEP (as gum xanthan) for two samples ranged from 95.3 to 108 and the relative standard deviations (R.S.D.s) were lower than 0.8% for gum xanthan solutions at concentrations of 1.0 and 1.5mugml(-1) (n=8). The results obtained for TEP in freshwater samples using the proposed spectrophotometric method and those obtained using a literature method are in agreement at the 95% confidence level and within an acceptable range of error.

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.

STRUCTURE OF THE CAPSULAR AND EXTRACELLULAR POLYSACCHARIDES PRODUCED BY THE DESMID SPONDYLOSIUM PANDURIFORME (CHLOROPHYTA)1

The filamentous desmid Spondylosium panduriforme (Heimerl) Teiling var. panduriforme f. limneticum (West & West) Teiling (Desmidiaceae), strain 072CH‐UFCAR, is surrounded by a well‐defined, mucilaginous capsule consisting of a capsular polysaccharide (CPS). This microalga also produces an extracellular polysaccharide (EPS), which can be isolated from the culture medium. Analysis of the carbohydrate composition of the two polymers by gas chromatography showed that they were different. Both were composed, of galactose, fucose, xylose, arabinose, rhamnose, and glucose but in different amounts. For example, glucuronic acid accounts for 24% of the EPS material but only traces were found in the CPS. Significant differences were also found during methylation analysis. Fucose appeared to have a higher degree of branching in the EPS than in the CPS. These branches were located on C‐3 and could be the position for the attachment of the glucuronic acid units in the EPS. The glucuronic acid was present as 1→4‐linked and terminal units. A possible explanation for the formation of the EPS is suggested.

Release of carbohydrates and proteins by a subtropical strain of Raphidiopsis brookii (cyanobacteria) able to produce saxitoxin at three nitrate concentrations.

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.

Toxicity of ionic copper to the freshwater microalga Scenedesmus acuminatus (Chlorophyceae, Chlorococcales)

A.T. Lombardi, T.M.R. Hidalgo, A.A.H. Vieira and A.L. Sartori. 2007. Toxicity of ionic copper to the freshwater microalga Scenedesmus acuminatus (Chlorophyceae, Chlorococcales). Phycologia 46: 74–78. DOI: 10.2216/06-40.1 Metal ecotoxicity testing with microalgae is subject to interferences such as change in bioavailable metal fraction by excreted metabolites. Moreover, high metal loads are usually necessary for obtaining 50% effect concentration (EC50) values based on cell number or chlorophyll a. Thus, sensitive parameters, which promptly respond to low dissolved metal variations at environmentally relevant concentrations, are much needed. The genus Scenedesmus, a coenobium-forming freshwater microalga, is cosmopolitan and inhabits pristine and contaminated waters. The present investigation addressed cellular phenotypic alterations in Scenedesmus acuminatus (Largerheim) Chodat 1902 caused by the presence of copper. Copper toxicity to the alga was investigated under laboratory-controlled conditions using a concentration range of 10−8 mol l−1 to 10−3 mol l−1 total dissolved Cu2+, thus including environmentally relevant concentrations. Free Cu2+ concentrations were determined using an ion-selective electrode calibrated with a metal ion buffer. The phenotypic plasticity of S. acuminatus was measured as the number of cells per coenobia and found to be readily affected at low copper concentrations. Under healthy conditions (10−8 mol l−1 total dissolved Cu2+, 10−11 mol l−1 free Cu2+) S. acuminatus formed eight-celled coenobia, whereas at 10−7 mol l−1 total dissolved Cu2+ (3 × 10−10 mol l−1 free Cu2+) these were absent and at 10−5 mol l−1 total dissolved Cu2+ (5 × 10−8 mol l−1 free Cu2+) free cells were predominant. Chlorophyll a synthesis was affected by 5 × 10−8 mol l−1 free Cu2+ or higher. EC50 (3.3 × 10−5 mol l−1 free Cu2+) was obtained using chlorophyll a values after 96-h exposure to the toxicant. The number of cells per coenobia was a more sensitive parameter than chlorophyll a to assess Cu toxicity, and at concentrations that may be encountered in the environment.

Activity of glycosidases from freshwater heterotrophic microorganisms on the degradation of extracellular polysaccharide produced by Anabaena spiroides (Cyanobacteria)

The activity of specific glycosidases during the degradation of the extracellular polysaccharide (EPS) produced by Anabaena spiroides was determined using MUF-substrates (MUF-monosaccharides). Polysaccharide degradation was found to occur in a two-phase process. The first consisted of high enzymatic activity that consumed 41% of the EPS at a relatively high rate, while the second consumed the remaining polysaccharide (59%) at a slower rate. A transition phase from the higher to the slower degradation rates was marked by a replacement of bacterial populations from coccoid to bacillus cells. During the degradation process, the bacterial biomass increased with the decrease of EPS, as revealed by bacterial cell counts. The enzymatic activity detected through the substrates MUF-a-D- and MUF-b-D-glucoside was higher than that detected by other substrates tested. The remaining glycosides were MUF-a-L-rhamnopyranoside, MUF-b-D-galactoside, MUF-a-D-mannopyranoside, MUF-b-D-fucoside, MUF-b-D-mannopyranoside, MUF-a-L-arabinopyranoside, and MUF-b-L-fucoside. The fluorescence emitted by each MUF-substrate was proportional to the concentration of the corresponding monosaccharide in A. spiroides EPS. This demonstrates the susceptibility of EPS produced by A. spiroides to enzymatic attack by bacterial populations.

An extracellular sulfated fucose-rich polysaccharide produced by a tropical strain of Cryptomonas obovata (Cryptophyceae)

A tropical strain of Cryptomonasobovata Skuja, isolated from a shallow oxbow lake,releaseda sulfated fucose-rich polysaccharide. The polysaccharide is composed mainly offucose (42%), N-acetyl-galactosamine (26%) and rhamnose (15%), with smallquantities of glucuronic acid, mannose, galactose, xylose and glucose. Sulfateaccounted for 1.7% total polysaccharide. Quantitative release was studied withcells exposed to optimal culture conditions contrasted with high irradiance andnitrate depletion. This latter set of conditions could simulate stresssituations usually found in the place from which this strain was isolated. Themonosaccharide composition of the polysaccharide was evaluated using PAD-HPLCand gas chromatography. The two irradiances tested (165 μmolm−2 s−1 and 2000 μmolm−2 s−1) had no significant effect onamounts of polysaccharide released by the cells. Differences were observed whenthe nitrate availability was varied. In the nitrate-depleted situation,extracellular polysaccharide production was 2.5 times higher than replete cellsafter 6 h at 165 μmol m−2s−1, and 2.25 times higher at 2000 μmolm−2 s−1.

Avaliação da potencialidade de microalgas dulcícolas como fonte de matéria-prima graxa para a produção de biodiesel

In this work, the fatty acid quantity and composition of six freshwater microalgae and soybean grains was determined by direct transesterification and gas chromatography analysis. The results showed that all the freshwater microalgae species presented a higher quantity of fatty acid than soybean grain. Choricystis sp. (A) provides 115% more fatty acids per gram of biomass than soybean grain. With regard to the fatty acid composition, Choricystis sp. (A) showed an adequate proportion of saturated and unsaturated fatty acids, with lower quantity of polyunsaturated fatty acids and, akin to some marine microalgae, constitutes an alternative raw material for biodiesel production.