Open Life Sci. 2015; 10: 52–60 Research Article Open Access Spyros Gkelis*, Pablo Fernández Tussy, Nikos Zaoutsos Isolation and preliminary characterization of cyanobacteria strains from freshwaters of Greece Abstract: Cyanobacterial harmful algal blooms (or 1 Introduction CyanoHABs) represent one of the most conspicuous waterborne microbial hazards. The characterization of Cyanobacteria are photosynthetic, prokaryotic organisms the bloom communities remains problematic because which occur primarily in freshwater and saline the cyanobacterial taxonomy of certain genera has not environments, but also in terrestrial ecosystems. Their yet been resolved. In this study, 29 planktic and benthic presence in lakes with high nutrient levels can lead to a cyanobacterial strains were isolated from freshwaters mass increase in cyanobacterial cell numbers, with the located in Greece. The strains were assigned to the genera formation of blooms, which results in a depreciation of Chroococcus, Microcystis, Synechococcus, Jaaginema, water quality [1]. Cyanobacterial harmful algal blooms Limnothrix, Pseudanabaena, Anabaena, and Calothrix (or CyanoHABs) represent one of the most conspicuous and screened for the production of the cyanotoxins waterborne microbial hazards to human and agricultural microcystins (MCs), cylindrospermopsins (CYNs), and water supplies, fishery production, and freshwater and saxitoxins (STXs) using molecular (PCR amplification of marine ecosystems [2]. This hazard results from the seven genes implicated in cyanotoxin biosynthesis) and production of cyanotoxins, harmful secondary metabolites, immunological (ELISA) methods. This study presents, which can have deleterious effects within reservoirs and in for the first time, a cyanobacteria culture collection downstream receiving water systems during releases [3]. from Greece, thus providing missing study material for In Greece, common bloom-forming cyanobacteria the understanding of bloom formation and cyanotoxin mainly belong to the genera Microcystis and Anabaena, production in the Mediterranean and for the polyphasic followed by Cylindrospermopsis and Aphanizomenon [1, characterization of important components of the 4, 5]. In addition to the bloom-forming cyanobacteria, phytoplankton. The combined use of molecular and a wide range of less abundant and lesser-known immunochemical methods allowed the identification of cyanobacteria, such as, filamentous (e.g. Pseudanabaena) MC producing strains, but further data are needed for CYN- or colonial (e.g. Aphanocapsa, Chroococcus, Cyanodictyon) and STX-producing cyanobacteria. The high percentage nanoplanktonic (2-20 μm) species [4] and Synechococcus- of MC-producing Microcystis strains in the urban Lakes type picocyanobacteria (<2 μm) [6] are present in blooms Kastoria and Pamvotis, frequently used for agriculture that rarely become dominant, but can represent an irrigation, fishing and recreation, highlights the potential important part of the total cyanobacterial biomass. risk for human health. Occasionally, benthic and/or periphytic cyanobacteria can be observed in phytoplankton. Keywords: Microcystis, Anabaena, Limnothrix, Calothrix, The characterization of the bloom communities’ cyanotoxins, molecular detection, lakes, ELISA structure remains problematic because the cyanobacterial taxonomy of certain genera has not yet been resolved [7]. Today, cyanobacterial diversity is examined using a DOI 10.1515/biol-2015-0006 polyphasic approach by assessing morphological and Received January 28, 2014; accepted August 21, 2014 molecular data (e.g. 8, 9), often combined with toxicological characters [10, 11]. The traditional cyanobacterial *Corresponding author: Spyros Gkelis: Department of Botany, classification [12-15] and the bacteriological classification School of Biology, Aristotle University of Thessaloniki, GR-541 24 [16] are based on morphological and genotypic (partial Thessaloniki, Macedonia, Greece, E-mail: [email protected] 16S rRNA gene sequences) data [9]. The comparison of Pablo Fernández Tussy, Nikos Zaoutsos: Department of Botany, morphological and genetic data is sometimes hindered School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Macedonia, Greece by the lack of cultures of several cyanobacterial © 2015 Spyros Gkelis et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Isolation and characterization of cyanobacteria strains 53 morphospecies and inadequate morphological data of Thessaloniki (AUTH) microalgae collection (Department of sequenced strains [8]. Furthermore, in order to evaluate the Botany, School of Biology) and can be accessed in http:// phenotypic plasticity within defined taxa, the variability cyanobacteria.myspecies.info/. observed in cultures has to be compared to the range in natural variation [17]. 2.3 Light microscopy The taxonomy of some of the potentially toxic cyanobacteria remains challenging [18], especially due to A Zeiss Axio imager z2 (Carl Zeiss, Germany) microscope the co-occurrence of several different morphotypes [7]. using bright field and differential interference contrast In Greece, cyanobacteria diversity and toxicity is mainly (EC Plan-Neofluar 5x/0,16,EC Plan-Neofluar 10x/0.3, Plan- known by field (e.g. [4, 5, 19]) and culture-independent Apochromat 20x/0.8, Plan-Neofluar 40x/0.75 DIC, Plan- 16S rRNA gene studies (e.g. [7]); only one publication [18] Neofluar 63x/1.25 Oil DIC, Plan-Neofluar 100x/1.30 Oil DIC) refers to Limnothrix cyanobacteria isolates. The objective was used. Microphotographs were taken with an Axio Cam of this paper is to isolate and characterize cyanobacteria MRc5 digital camera (Carl Zeiss, Germany). from freshwaters of Greece, with respect to their ability to produce cyanotoxins. 2.4 Identification The strains were identified to the species or genus level 2 Experimental Procedures according to Anagnostidis & Komárek [12, 13), Komárek & Anagnostidis (14, 15, 23, 24), Castenholz [16], taking into 2.1 Growth media and growth conditions consideration current taxonomic status [17]. Solid growth medium: agar plates 53 and 90 mm in 2.5 DNA extraction and PCR analyses diameter containing BG-11 media [20] with or without (for the nitrogen-fixing strains) nitrogen, 1.2% w/v [21] agar In order to identify toxic strains, different primer pairs, (Sigma-Aldrich, Germany). Liquid growth medium: BG-11 previously described in the literature, were used to with or without nitrogen in 100, 250 and 500 mL culture detect different gene targets known to be involved in the flasks. biosynthesis of either MC, CYN or STX. DNA was extracted Cultures were grown as liquid batch cultures at 20±2oC using the protocol described in Atashpaz et al. [25] for Gram or 25±1oC (for Microcystis) at a photosynthetic photon negative bacteria. PCR was carried out on the DNA extracts flux density of 20 μmol m-2 s-1 provided by cool white light using the primer pairs shown in Table 2 and PCR conditions fluorescent lamps (Sylvania Standard F36W/154-T8, SLI) in described in detail by Gkelis & Zaoutsos [5]. Thermal a 16:8 h light:dark cycle. cycling was carried out using an Eppendorf MasterCycler Pro (Eppendorf). PCR products were separated by 1.5% 2.2 Sampling Sites and Strain isolation (w/v) agarose gel in 1X TAE buffer. The gels were stained with ethidium bromide and photographed under UV Strains were isolated from surface water samples collected transillumination. from freshwaters of Greece between 1999 and 2010 (Table DNA extracted from Microcystis aeruginosa M6 strain 1); for a description of the Kerkini Reservoir and Lakes was used as positive control for the amplification of mcyA, Amvrakia, Doirani, Kastoria, Mikri Prespa, Pamvotis, mcyB and mcyE gene targets; DNA from Cylindrospermopsis Paralimni, Volvi, see [1 and 4]. Lake Pikrolimni is located raciborskii Aqs strain was used as positive control for in the basin of Kilkis plain, near Thessaloniki (23 km), in the amplification of the ps (peptide syntethase) and pks northern Greece. It is a small, shallow lake which usually (polyketide synthase) genetic determinants; DNA from dries out during summer. It has an average depth of Aphanizomenon gracile A040 strain was used as positive about 0.5–0.7 m and covers an area about 4.5 km2 when control for the detection of sxtI target gene (see [26]). All it is flooded [22]. Strains were isolated on solid growth positive controls we used produced an amplification media using classical microbiological techniques and product under the tested conditions. grown as batch clonal unialgal cultures. The strains were purified unialgal by repeated transfer of single colonies or 2.6 Cyanotoxin analyses trichomes of cyanobacteria on BG-11 medium agar plates; all strains were derived from a single colony or trichome. The Abraxis Microcystin (520011), Saxitoxin (52255B), and The isolates were deposited in Aristotle University of Cylindrospermopsin (522011) Microtiter Plate Kits were 54 S. Gkelis et al. used to determine the presence of Microcystins (MCs), percentage of >15% were not accepted. Strains were Saxitoxins (STXs), and Cylindrospermopsins (CYNs), considered positive for a cyanotoxin when concentration respectively. Eight-to-ten mL from each culture were was higher of the lowest concentration of the standards centrifuged and the pellet was freeze-dried. provided for each cyanotoxin. MC, STX and CYN from each strain were extracted by placing up to 1200mg of freeze-dried material in eight mL of water