Growth of Microcystisstrains isolated from environments with the presence and absence of submerged macrophytes in coexistence with Ceratophyllum demersum

Cyanobacterial blooms can cause severe ecological and health problems in drinking water reservoirs. To alleviate this problem, allelopathically active submerged macrophytes can be used to reduce cyanobacterial growth. Accordingly, this study aimed to evaluate the sensitivity of strains of the Microcystis aeruginosacomplex isolated from reservoirs with the presence and absence of submerged macrophytes to the allelochemicals of Ceratophyllum demersum.A coexistence experiment was carried out between the submerged macrophyte C. demersum and four Microcystisstrains, with two treatments for each strain, one in coexistence with the submerged macrophyte (7 g L-1) and control (in the absence of the macrophyte). Two strains of M. aeruginosa(BMIUFRPE-06 and BMIUFRPE-07) and two of M. panniformis(BMIUFRPE-08 and BMIUFRPE-09) were used, which were isolated from Cajueiro (with submerged macrophytes) and Tapacurá (without submerged macrophytes) reservoirs, respectively. The biomass of Microcystisstrains from the reservoir without macrophytes (BMIUFRPE-08 and BMIUFRPE-09) was significantly inhibited in 96% (T-test: p 0.05; growth rate -ANOVA: p > 0.05). These results suggest that strains isolated from environments with submerged macrophytes are less sensitive to allelochemicals of these plants,as these strains may be adapted to the coexistence with submerged macrophytes.

Microcystis Chemotype Diversity in the Alimentary Tract of Bigheaded Carp.

Most cyanobacterial organisms included in the genus Microcystis can produce a wide repertoire of secondary metabolites. In the mid-2010s, summer cyanobacterial blooms of Microcystis sp. occurred regularly in Lake Balaton. During this period, we investigated how the alimentary tract of filter-feeding bigheaded carps could deliver different chemotypes of viable cyanobacteria with specific peptide patterns. Twenty-five Microcystis strains were isolated from pelagic plankton samples (14 samples) and the hindguts of bigheaded carp (11 samples), and three bloom samples were collected from the scums of cyanobacterial blooms. An LC-MS/MS-based untargeted approach was used to analyze peptide patterns, which identified 36 anabaenopeptin, 17 microginin, and 13 microcystin variants. Heat map clustering visualization was used to compare the identified chemotypes. A lack of separation was observed in peptide patterns of Microcystis that originated from hindguts, water samples, and bloom-samples. Except for 13 peptides, all other congeners were detected from the viable and cultivated chemotypes of bigheaded carp. This finding suggests that the alimentary tract of bigheaded carps is not simply an extreme habitat, but may also supply the cyanobacterial strains that represent the pelagic chemotypes.

KMnO4-Fe(II) pretreatment to enhance Microcystis aeruginosa removal by aluminum coagulation: Does it work after long distance transportation?

KMnO4-Fe(II) pretreatment was proposed to enhance Microcystis aeruginosa (M. aeruginosa) removal by aluminum (Al) coagulation in drinking water treatment plants (DWTPs) in our previous study. This study aims to optimize this process and evaluate the feasibility of using the process at water sources, which are usually far away from DWTPs. The optimum molar ratio of KMnO4 to Fe(II) [Formula: see text] is observed to be 1:3 with respect to algae removal and residual manganese (Mn) control. As indicated from flow cytometer analysis, KMnO4 at <20 µM promisingly maintains cell integrity, with damaged cell ratios of below 10%. KMnO4 at 30 and 60 µM damages M. aeruginosa cells more significantly and the damaged cell ratios increase to 21% and 34% after 480 min. The intracellular organic matter (IOM) release can be controlled by the subsequent introduction of Fe(II) to quench residual KMnO4. KMnO4-Fe(II) pretreatment at the KMnO4 dose of 10 µM dramatically enhances the algae removal by over 70% compared to that by Al coagulation, even if KMnO4 and Fe(II) are introduced 480 min prior to the addition of Al2(SO4)3. The Al doses can be reduced by more than half to achieve the same algae removal. Furthermore, the deposition of the tiny Fe-Mn precipitates formed rarely occurs, as indicated by a settleability evaluation prior to Al addition. The KMnO4-Fe(II) process can be sequentially dosed at intake points in water sources to achieve moderate inactivation of algae cells and to enhance algae removal in DWTPs thereafter.

Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 g.L1 to 0.05 g.L1. Besides, an increase in the bacterial countfrom 71×105 bacteria.mL1 to 117×105 bacteria.mL1was observed along the incubation. Conclusions The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacteriumof the genus Pseudomonasthat can degrade [D-Leu1] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.

Human and rat hepatocyte toxicity and protein phosphatase 1 and 2A inhibitory activity of naturally occurring desmethyl-microcystins and nodularins.

Contamination of water, foods and food supplements by various genera of cyanobacteria is a serious health problem worldwide for humans and animals, largely due to the toxic effects of microcystins (MCs) and nodularin (NOD), a group of hepatotoxic cyclic peptides. The toxins occur in variable structures resulting in more than 90 different MCs and 8 different NODs, many of them not having been investigated for their toxic potency. Potent MCs such as MC-LR have been shown to elicit their hepatotoxic potency via inhibition of hepatic protein phosphatases (PP) 1 and 2A leading to over-phosphorylation of vital cellular proteins. This mechanism of action is also thought to be responsible for the long term tumor promoting action of certain MCs and NOD in the liver. Here, we report on the isolation of certain MCs and NOD as well as a number of their desmethylated derivatives from algae bloom. Subsequently, we determined the cytotoxicity of these compounds in isolated primary human and rat hepatocytes in culture. In parallel experiments, we analyzed the inhibitory potency of these congeners on PP1 and 2A using commercially available enzymes. We found in primary rat hepatocytes that MC-LR, -YR and NOD were cytotoxic, namely in the 10 to >50 nM range, while MC-RR was not. The desmethylated congeners of MC-LR, -YR, and NOD were equally or more-toxic as/than their fully methylated counterparts. In primary human hepatocytes we could show that MC-LR, NOD and the desmethylated variants [³Asp]MC-LR, [7Dha]MC-LR and [¹Asp]NOD were cytotoxic in the 20 to >600 nM range. Inhibition data with human, bovine and rabbit protein phosphatases 1 and 2A were roughly in accordance with the cytotoxicity findings in human and rat hepatocytes, i.e. desmethylation had no pronounced effects on the inhibitory potencies. Thus, a variety of naturally occurring desmethylated MC and NOD congeners have to be considered as being at least as toxic as the corresponding fully methylated derivatives.

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Recreational exposure to microcystins during algal blooms in two California lakes.

We conducted a study of recreational exposure to microcystins among 81 children and adults planning recreational activities on either of three California reservoirs, two with significant, ongoing blooms of toxin-producing cyanobacteria, including Microcystis aeruginosa (Bloom Lakes), and one without a toxin-producing algal bloom (Control Lake). We analyzed water samples for algal taxonomy, microcystin concentrations, and potential respiratory viruses (adenoviruses and enteroviruses). We measured microcystins in personal air samples, nasal swabs, and blood samples. We interviewed study participants for demographic and health symptoms information. We found highly variable microcystin concentrations in Bloom Lakes (<10 microg/L to >500 microg/L); microcystin was not detected in the Control Lake. We did not detect adenoviruses or enteroviruses in any of the lakes. Low microcystin concentrations were found in personal air samples (<0.1 ng/m(3) [limit of detection]-2.89 ng/m(3)) and nasal swabs (<0.1 ng [limit of detection]-5 ng). Microcystin concentrations in the water-soluble fraction of all plasma samples were below the limit of detection (1.0 microg/L). Our findings indicate that recreational activities in water bodies that experience toxin-producing cyanobacterial blooms can generate aerosolized cyanotoxins, making inhalation a potential route of exposure. Future studies should include collecting nasal swabs to assess upper respiratory tract deposition of toxin-containing aerosols droplets.

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR.

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.

Peptide diversity in strains of the cyanobacterium Microcystis aeruginosa isolated from Portuguese water supplies.

Strains of the cyanobacterium Microcystis aeruginosa were isolated into pure culture from a variety of lakes, rivers, and reservoirs in Portugal. Samples were tested with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to investigate the presence of various peptide groups including aeruginosins, microginins, anabaenopeptins, cyanopeptilins, microcystins, and microviridins and other peptide-like compounds. Binary data, based on the presence and absence of different peptide groups, were analyzed by phylogenetic inference. DNA was also extracted from the samples and tested using a range of primers. Those strains that gave positive results for a Microcystis-specific primer pair were further analyzed for the presence of genes linked to the biosynthesis of microginin and microcystin. The results showed that a wide range of microcystin forms were produced by the strains among which MC-LR, -FR, -RR, -WR, and -YR were the most common. The peptide profiles obtained from the MALDI analysis were assessed using cluster analysis which resulted in the formation of distinct groups or chemotypes.

[Effect of toxins of cyanobacteria of the Microcystis genus from the Shershnevo Reservoir on DNA, cell cycle, and apoptosis of bone marrow cells in CBA mice].

The effect of toxins of a Cyanobacterium sample of the Shershnevo Reservoir on DNA, which was presented by cyanobacteria of the Microcystis genera, on the bone marrow of male CBA mice (whose age was 3 months and weight 24 g) was evaluated. With intraperitoneal administration, LD50 and LD16 of this sample for male CBA mice were 48.4 and 42.1 mg/kg, respectively. Administration of Microcystis cyanobacterial sample from the Shershnevo Reservoir in doses of 1/10 of LD16 and 1/2 of LD16, and LD16 was found to cause a dose-dependent reduction in the number of bone marrow nucleated cells, a dose-dependent increase in the rate of cell apoptotic death, a reduction in the duration of a cell cycle (within the first 12 hours), which gave way to an increase in the duration of the cycle 24 hours after administration, a dose-dependent increase in the frequency of micronuclei in the murine bone marrow eryphrocytes, and a dose-dependent decrease in the polychromatophil/normochromatophil ratio in the murine bone marrow.

Recreational exposure to low concentrations of microcystins during an algal bloom in a small lake.

We measured microcystins in blood from people at risk for swallowing water or inhaling spray while swimming, water skiing, jet skiing, or boating during an algal bloom. We monitored water samples from a small lake as a Microcystis aeruginosa bloom developed. We recruited 97 people planning recreational activities in that lake and seven others who volunteered to recreate in a nearby bloom-free lake. We conducted our field study within a week of finding a 10-microg/L microcystin concentration. We analyzed water, air, and human blood samples for water quality, potential human pathogens, algal taxonomy, and microcystin concentrations. We interviewed study participants for demographic and current health symptom information. Water samples were assayed for potential respiratory viruses (adenoviruses and enteroviruses), but none were detected. We did find low concentrations of Escherichia coli, indicating fecal contamination. We found low levels of microcystins (2 microg/L to 5 microg/L) in the water and (<0.1 ng/m(3)) in the aerosol samples. Blood levels of microcystins for all participants were below the limit of detection (0.147 microg/L). Given this low exposure level, study participants reported no symptom increases following recreational exposure to microcystins. This is the first study to report that water-based recreational activities can expose people to very low concentrations of aerosol-borne microcystins; we recently conducted another field study to assess exposures to higher concentrations of these algal toxins.

[Critical collapse pressure of gas vesicles in six strains of cyanobacteria].

Two methods including filtration (vacuity is 0.02 MPa) and centrifugation ( < 500 r/min) were applied to concentrate algal biomass, and capillary compression tube and the concentrated algae suspensions, critical pressure distribution of gas vesicles in six cyanobacteria were also investigated. The results showed that unicellular Microcystis aeruginosa cells couldn't be concentrated effectively by filtration or centrifugation, but colony of Microcystis wesenbergii and Microcystis flosaquae, and filament of Planktothrix mougeotii could be concentrated by centrifugation and filtration respectively. Both filtration and centrifugation had a negligibly impact on gas vesicles content of cells, and the loss of gas vesicles was lower than 7%. The mean critical pressure values of five strains Microcystis were very close, which ranged from 0.64 to 0.67 MPa, and the mean critical pressure of P. mougeotii was 0.715 MPa. All of the six cyanobacteria were isolated from three shallow lakes, and their mean critical pressure values were lower than those cyanobacteria that inhabit in deep lakes or reservoirs. At the same condition of illumination density and temperature, turgor pressures of two unicellular Microcystis were higher than those of colonial Microcystis.

Integration of dissolved gas flotation and nanofiltration for M. aeruginosa and associated microcystins removal.

The removal of Microcystis aeruginosa and associated microcystins was investigated by a dissolved gas flotation (preceded by coagulation/flocculation)-nanofiltration (NF) sequence. The experiments were conducted with a freshwater spiked with M. aeruginosa cell aggregates to simulate a naturally occurring bloom. Two types of gases were used in the flotation pre-treatment, air (DAF) and a mixture of CO(2)/air. Very good results in terms of NF fluxes, overall removal efficiencies and final water quality were achieved with both sequences. However, the CO(2)/air mixture presented no benefit to the overall sequence, both in terms of toxin release to water during flotation and lower natural organic matter removal by NF, which was due to an overall negative effect of the acid pH. NF was able to completely remove cyanobacteria (100% removal efficiency of chlorophyll a) and microcystins (always under the quantification limit), regardless of the pre-treatment used and the water recovery rate (up to 84%). Therefore, DAF-NF sequence is a safe barrier against M. aeruginosa and microcystins in drinking water. In addition, it ensures an excellent control of particles, disinfection by-products formation, and other micropollutants that may be present in raw water.

Occurrence of toxin-producing cyanobacteria blooms in a Brazilian semiarid reservoir

Nós relatamos a ocorrência de florescimentos de cianobactérias e a presença de cianotoxinas em amostras de água do reservatório Armando Ribeiro Gonçalves (06º 08Æ S; 37º 07Æ W) situado no Estado do Rio Grande do Norte, na região semi-árida do Brasil. Cianobactérias foram identificadas e quantificadas nos períodos seco e chuvoso do ano 2000. Cianotoxinas tais como, microcistinas, saxitoxinas e cilindrospermopsinas foram quantificadas por HPLC e ELISA. Florescimentos tóxicos mistos de Cylindrospermopsis raciborskii, Microcystis spp (M. panniformis, M. protocystis, M. novacekii) e Aphanizomenon ssp (Aphanizomenon gracile, A. cf. manguinii, A. cf. issastschenkoi) foram persistentes e representaram 90-100% da comunidade fitoplanctônica ao longo do período estudado. No período de chuvas, florescimentos tóxicos de Cylindrospermopsis raciborskii coincidiram com maiores valores de saxitoxinas (3,14 µg.L-1). Entre o período de chuva e estiagem, ocorreram florescimentos tóxicos de Microcytis spp, excedendo o valor mínimo aceitável para consumo humano (8,8 µg.L-1). Na estiagem, baixas concentrações de saxitoxinas foram detectadas em florescimentos menos intensos com co-dominância de Cylindrospermopsis raciborskii, Microcystis spp e Aphanizomenon spp. Nossos resultados revelaram a presença de microcistinas (8,8 µg.L-1) e saxitoxinas (3,14 µg.L-1) na água bruta, a partir da segunda quinzena de abril até o final de maio de 2000. A ocorrência de blooms tóxicos de cianobactérias no reservatório em estudo aponta um risco permanente de cianotoxinas em águas de abastecimento e indica a necessidade da implementação de medidas de controle das florações, visando à melhoria da qualidade da água. A exposição das populações locais às cianotoxinas, pela sua potencial acumulação em musculatura de peixes, também deve ser considerada.

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river.

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.