Hanna Mazur-Marzec

The effect of salinity on the growth, toxin production, and morphology of Nodularia spumigena isolated from the Gulf of Gdańsk, southern Baltic Sea

Culture experiments on the toxic Nodularia spumigena strain NSGG-1 isolated from the Gulf of Gdańsk showed a significant effect of salinity on growth and nodularin production. Growth of the NSGG-1 strain, was optimal between 7 and 18 psu, lower at 3 and 24 psu and was significantly inhibited at the extreme salinities of 0 and 35 psu. Nodularin (NOD) content of N. spumigena, estimated by the NOD/Chla ratios, correlated positively with salinity and increased from 0 to 35 psu. The NOD/Chla ratio on day 10 of growth was high, and, reached the maximum at day 30. A sudden increase in salinity from 7 to 18 and 35 psu resulted in plasmolysis of Nodularia cells. Salinity was also observed to have other effects on NSGG-1; the filaments were longest at 7 psu, while an increased number of akinetes were formed at 35 psu. The number of heterocytes was markedly reduced at the extreme salinities. This latter finding might explain why Nodularia blooms do not occur outside a certain salinity range in nitrogen-deficient waters.

Cyanobacteria and cyanotoxins in Polish freshwater bodies

In this work, the authors examined the presence of cyanobacteria and cyanotoxins in 21 samples collected from fresh water bodies located in 5 provinces in Poland: Lublin (2), Podlasie (1), Pomerania (6), Warmia-Masuria (1) and Wielkopolska (11). In addition, to determine the general pattern of geographical distribution, frequency of cyanobacteria occurrence, and cyanotoxins production, the published data from 238 fresh water bodies in Poland were reviewed. On the basis of these collected results, we concluded that Planktothrix, Aphanizomenon, Microcystis and Dolichospermum were dominant. The general pattern in geographical distribution of the identified cyanobacterial genera was typical of other eutrophic waters in Europe. The production of cyanotoxins was revealed in 18 (86%) of the 21 samples analyzed in the present work and in 74 (75%) of the 98 total water bodies for which the presence of toxins had been examined. Among the 24 detected microcystin variants, [Asp3]MC-RR was most common. These results can be verified when more data from the less explored water bodies in the southern and eastern parts of Poland are available.

The effect of cyanobacterial blooms in the Siemianówka Dam Reservoir on the phytoplankton structure in the Narew River

The effect of cyanobacterial blooms on the phytoplankton structure in the lowland Narew River (north-eastern Poland) was examined. The studies were carried out at stations situated at different distances from the eutrophic Siemianówka Dam Reservoir. In 2008, the investigated lowland reservoir and the outflowing river were characterized by the dominance of toxic cyanobacteria (from July to October). At a station situated 130 km below the dam, species composition in the river was very similar to that in the shallow reservoir. Planktothrix agardhii was the main and permanent dominant, both in limnoplankton and potamoplankton. The current study indicates that the eutrophic Siemianówka Dam Reservoir is the main and rich source of phytoplankton for the outflowing Narew River. Cyanobacteria were dominant in the river phytoplankton at all sampling stations, but their share in phytoplankton biomass gradually decreased with the distance from the dam. Chemical analysis revealed the presence of microcystins (MCs) in water samples collected from June to October. The toxins, mainly demethylated MC analogues, were detected at a long distance down the river, even 100 km from the dam. Maximum concentration of MCs (14.3 μg l−1) was measured on 13 October, 9.1 km below the dam.

Diversity of Peptides Produced by Nodularia spumigena from Various Geographical Regions

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.

Biodegradation of nodularin and effects of the toxin on bacterial isolates from the Gulf of Gdańsk

Nodularin (NOD), a cyclic pentapeptide produced by the cyanobacterium Nodularia spumigena, is one of the most abundant natural metabolites occurring in the Baltic Sea. The present study investigated the role of this compound in the interactions between cyanobacteria and other bacteria. The toxin inhibited the growth of 15 out of 32 bacterial strains isolated from water and sediments of the Gulf of Gdańsk, southern Baltic Sea. Most of the bacteria sensitive to NOD belonged to the Proteobacteria phylum. Incubation of nodularin in the presence of the bacterial isolates did not reveal any NOD-degrading activity. However, natural microbial communities from sediment removed the toxin within 5-7 days. Analysis by liquid chromatography/hybrid quadrupole-time-of-flight mass spectrometry with turboion spray (QTOF-LC/MS/MS) revealed seven biodegradation products, including five novel ones. The results showed that not only freshwater microorganisms, but also those living in brackish waters, play an important role in cyanotoxin removal.

Cyanobacterial hepatotoxins, microcystins and nodularins, in fresh and brackish waters of the Pomeranian Province, Northern Poland

Cyanobacterial hepatotoxins, microcystins and nodularins, in fresh and brackish waters of the Pomeranian Province, northern Poland Microcystins (MCs) and structurally related nodularins (NODs) are hepatotoxic cyclic peptides produced by bloom-forming cyanobacteria. These toxins have been implicated in the deaths of wild and domestic animals as well as in incidents of human illness. Cyanobacterial toxins occurring in the fresh and brackish waters of the Pomeranian Province, northern Poland were characterized in this study. Water samples collected from seven lakes in August and September 2005 were analysed by high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA) and protein phosphatase inhibition assay (PPIA). Cyanobacterial toxins present in field samples and in an isolated strain of Planktothrix agardhii were also characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In most of the fresh water samples MC-LR, MC-RR and MC-YR dominated. In the lakes where P. agardhii was most abundant demethylated microcystin variants tentatively identified as [D-Asp3]MC-LR, [D-Asp3]MC-YR and [D-Asp3]MC-RR, were found. Total concentrations of the toxins measured by HPLC ranged from 0.1 μg 1-1 to 305.4 μg 1-1. Nodularia spumigena bloom samples were collected from brackish waters of the Gulf of Gdańsk, southern Baltic, and LC-ISP-MS/MS of extract from these revealed the presence of two geometrical isomers of linear nodularin and nodularin variant with aspartic acid methyl ester [MeAsp1(OMe)]NOD.

Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria.

Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Baltic cyanobacteria – a source of biologically active compounds

Cyanobacteria are effective producers of bioactive metabolites, including both acute toxins and potential pharmaceuticals. In the current work, the biological activity of 27 strains of Baltic cyanobacteria representing different taxonomic groups and chemotypes were tested in a wide variety of assays. The cyanobacteria showed strain-specific differences in the induced effects. The extracts from Nodularia spumigena CCNP1401 were active in the highest number of tests, including protease and phosphatase inhibition assays. Four strains from Nostocales and four from Oscillatoriales increased proliferation of mitogen-stimulated human T cells. In antimicrobial assays, Phormidium sp. CCNP1317 (Oscillatoriales) strongly inhibited the growth of six fouling Gammaproteobacteria. The growth of monocotyl Sorghum saccharatum was inhibited by both toxin-producing and ‘non-toxic’ strains. The Baltic cyanobacteria were also found to be a rich source of commercially important enzymes. Among the 19 enzymatic activities tested, alkaline phosphatase, acid phosphatase, esterase (C4 and C8), and naphthol-AS-BI-phosphohydrolase were particularly common. In the cyanobacterial extracts, different peptides which may have been responsible for the observed effects were identified using LC-MS/MS. Their structures were classified to microcystins, nodularins, anabaenopeptins, cyanopeptolins, aeruginosins, spumigins and nostocyclopeptides.

Increased risk of exposure to microcystins in the scum of the filamentous cyanobacterium Aphanizomenon flos-aquae accumulated on the western shoreline of the Curonian Lagoon.

Concentration of toxic cyanobacteria blooms on the downwind shore of high recreational amenity water bodies with largely increases the risk of exposure to cyanotoxins. In this study analysis of phytoplankton structure, cyanotoxin composition and concentration was performed on cyanobacteria scum material, high- and low-density bloom samples in the Curonian Lagoon. We found that the concentration of cyanotoxins in the scum material increased from ∼30 to ∼300-fold compared to bloom samples. In Microcystis aeruginosa dominated samples microcystin-LR was present at the highest concentration, while the dominance of Planktothrix agardhii was associated with the occurrence of dmMC-RR as the major microcystin variant. The toxicological potential of cyanobacterial scums in the Curonian Lagoon is emphasized, and management by removal of these scums is proposed.