Justyna Kobos

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.

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.

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.

Non-ribosomal peptides produced by Planktothrix agardhii from Siemianówka Dam Reservoir SDR (northeast Poland)

Planktothtrix agardhii (Oscillatoriales) is a filamentous cyanobacterium, which frequently forms blooms in shallow, polymictic and eutrophicated waters. This species is also a rich source of unique linear and cyclic peptides. In the current study, the profile of the peptides in samples from the P. agardhii-dominated Siemianówka Dam Reservoir (SDR) (northeast Poland) was analyzed for four subsequent years (2009–2012). The LC–MS/MS analyses revealed the presence of 33 peptides. Twelve of the most abundant ones, including five microcystins, five anabaenopeptins, one aeruginosin and one planktocyclin, were present in all field samples collected during the study. The detection of different peptides in two P. agardhii isolates indicated that the SDR population was composed of several chemotypes, characterized by different peptide patterns. The total concentration of microcystins (MCs) positively correlated with the biomass of P. agardhii. Between subsequent years, the changes in the ratio of the total MCs concentration to the biomass of P. agardhii were noticed, but they were less than threefold. This is the first study on the production of different classes of non-ribosomal peptides by freshwater cyanobacteria in Poland.

The potential causes of cyanobacterial blooms in Baltic Sea estuaries

The potential causes of cyanobacterial blooms in Baltic Sea estuaries Nodularia spumigena Mertens, Aphanizomenon flos-aquae (L.) Ralfs and some species of the genus Anabaena are the dominant cyanobacterial taxa occurring in the Gulf of Gdańsk. The heterocystous cyanobacteria use dissolved molecular N2 as an additional nitrogen source, and this allows them to bloom during the summer when growth of other phytoplankton species is normally nitrogen-limited. Although cyanobacterial blooms have been reported in the Baltic Sea since the mid-19th century, the extent and intensity of blooms have recently increased due to anthropogenic sources of eutrophication. Increased river phosphorus input and changes in the phosphorus to nitrogen ratio are implicated as causal factors. After us the initial cause of the cyanobacterial bloom is a low N:P ratio, which indicates phosphorus excess, i.e. favourable nutrient conditions for nitrogen-fixing algae. An N:P ratio of 10 has been considered an approximate value for the N:P requirements of Baltic phytoplankton. For several years this ratio has been lower than 10. The mean annual value of the N:P ratio for the water of the Gulf of Gdańsk ranged from 3 to 7. Differences in the intensity of blooms observed in different years could be linked to temperature. During hot summers, when the seawater temperature increased to 20°C, large blooms were noted. For the cyanobacterial blooms in the Baltic Sea, the low N:P ratio is the primary factor and high temperature is a starting point.

The use of fosmid metagenomic libraries in preliminary screening for various biological activities

BackgroundIt is generally believed that there are many natural sources of as yet unknown bioactive compounds with a high biotechnological potential. However, the common method based on the use of cell extracts in the preliminary screening for particular molecules or activities is problematic as amounts of obtained compounds may be low, and such experiments are hardly reproducible. Therefore, the aim of this work was to test whether a novel strategy to search for previously unknown biological activities can be efficient. This strategy is based on construction of metagenomic libraries and employment of Escherichia coli strains as cell factories producing compounds of properties potentially useful in biotechnology.ResultsThree cyanobacterial metagenomic libraries were constructed in the fosmid system. The libraries were screened for various biological activities. Extracts from selected E. coli clones bearing constructs with fragments of cyanobacterial genomes revealed antimicrobial or anticancer activities. Interestingly, stimulation of growth of host bacteria bearing particular plasmids with certain cyanobacterial genes was detected, suggesting a potential possibility for improvement of E. coli cultivation during biotechnological production. The most interesting plasmids were sequenced, and putative mechanisms of biological effects caused by cyanobacterial gene products are discussed.ConclusionsThe strategy of exploring cyanobacteria as sources of bioactive compounds, based on E. coli cell factories producing compounds due to expression of genes from metagenomic libraries, appears to be effective.

Distribution of invasive Cylindrospermopsis raciborskii in the East-Central Europe is driven by climatic and local environmental variables

Mechanisms behind expansion of an invasive cyanobacterium Cylindrospermopsis raciborskii have not been fully resolved, and different hypotheses, such as global warming, are suggested. In the East-Central Europe, it is widely occurring in western part of Poland but only in single locations in the East due to some limiting factors. Therefore, broad-scale phytoplankton survey including 117 randomly selected lakes in Poland and Lithuania was conducted. The results showed that C. raciborskii occurred widely in western part of Poland but was absent from other regions and Lithuania except one lake. The regions in which C. raciborskii was present had higher annual mean air temperature, higher maximum air temperature of the warmest month and higher minimum temperature of the coldest month, demonstrating that average air temperature, and indirectly, the duration of growing season might be more important factor driving C. raciborskii distribution than measured in situ water temperature. In turn, the presence of C. raciborskii in single localities may be more related to physiological adaptations of separated ecotype. Collectively, these results provide novel evidence on the influence of temperature on C. raciborskii distribution in East-European regions but also indicate high ecological plasticity of this species.

Simple screening technique for determination of adsorbed and absorbed mercury in particulate matter in atmospheric and aquatic environment

The threat connected to mercury results from its capacity to be transported over long distances and its ability to bioaccumulate and biomagnify in the trophic chain, making it a global problem. Humans are situated at the top of the trophic ladder, and excess mercury manifests itself mainly in the onset of neurological conditions. The toxicity of mercury, as well as its residence time, depends on the form in which it occurs. However, analysis of mercury speciation is time-consuming and poses a high risk of additional or negative contamination. Hence, the mercury thermodesorption method, and particularly its use for fractionating Hg, offers many new possibilities. Here, the thermodesorption technique was applied to the determination of mercury fraction in particulate matter using a DMA-80 direct mercury analyser (Milestone, Italy). The presented method allows direct (without prior mineralisation) determination of labile and stabile mercury fractions within a relatively short time. Heating sample in subsequent temperatures enables determination the share of mercury adsorbed on the surface (mainly associated with halogenides (Hgads1) and HgSO4/HgO/HgF2 (Hgads2), as well as absorbed within the suspended particulate organic matter (Hgabs), in a relatively short time. This fractionation is of great importance in terms of estimating the transfer of mercury to and along the trophic chain. This method determines the contribution of two stable mercury fractions:: HgS and residual Hg, strongly bound to particulate matter matrix (Hgres). The novelty of this technique is also its joint ability to determine gaseous mercury bound to airboirne particulate matter, which will enable better understand Hg cycling in the atmosphere as well as mercury fraction in dry deposition flux. This method enables assessment of global mercury circulation in environment.

Mercury in the Diatoms of Various Ecological Formations

Mercury is a neurotoxin, its main source in the human organism being fish and seafood. The first level in the marine food web is formed of planktonic and benthic photosynthetic microorganisms, which form a biofilm on the surface of the hard bottom (epilithon) or plants (epiphyton). They are carriers of nutritional as well as toxic substances and pass these on to subsequent levels of the trophic web. Their biomass is often dominated by diatoms. This was the basis for the presented study into Hg accumulation in epilithic, epiphytic and planktonic diatoms, which was carried out in 2012–2013 in the coastal zone of the Puck Lagoon and the Gulf of Gdańsk (southern Baltic). In this coastal area, both micro- and macroorganisms develop particularly intensively. The collected results indicate an increase in Hg concentration in the biofilm during the warm season which, with the lengthening of the vegetative period due to global warming in recent years, is of great significance. As a consequence, the annual mercury load entering the trophic web is larger in comparison with a year in which there is a long, cold winter. An important parameter influencing the accumulation of Hg was the function of those organisms from the biofilm-forming communities. In this case, the highest concentrations of Hg were measured in organisms forming high-profile guilds.