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Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

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Abstract

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.

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References

  • Aboal, M., M. A. Puig & A. D. Asencio, 2005. Production of microcystins in calcareous Mediterranean streams: the Alharabe River, Segura River basin in south-east Spain. Journal of Applied Phycology 17: 231–243.

    Article  CAS  Google Scholar 

  • Allen, M. M., 1968. Simple conditions for growth of unicellular blue-green algae on plates. Journal of Phycology 4: 1–4.

    Article  CAS  Google Scholar 

  • Altschul, S. F., W. Gish, M. Miller, E. W. Myers & D. J. Lipman, 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403–410.

    PubMed  CAS  Google Scholar 

  • Amé, V., R. Echenique, S. Pflugmacher & A. Wunderlin, 2006. Degradation of microcystin-RR by Sphingomonas sp. CBA4 isolated from San Roque reservoir (Córdoba – Argentina). Biodegradation 17: 447–455.

    Article  CAS  Google Scholar 

  • ANZECC & ARMCANZ, 2000. Australian and New Zealand guidelines for fresh and marine water quality; Australian and New Zealand Environmental and Conservation Council (AZECC) and Agriculture and Resource Management Council of Australia and New Zealand (ARMCANZ), Canberra [http://www.rpdc.tas.gov.au/soer/source/718/index.php].

  • APHA (American Public Health Association). American Water Works Association, and Water Pollution Control Federation, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, Washington, DC.

    Google Scholar 

  • Azevedo, S. M. F. O. & V. F. Magalhães, 2002. Causes and consequences of the presence of toxic cyanobacteria in Brazilian ecosystems. In Sar, E. A., M. E. Ferrario & B. Reguera (eds), Harmful Algal Blooms in South America. Spanish Institute of Oceanography, Madrid, Spain: 226–228.

    Google Scholar 

  • Birnboim, H. C. & J. Doly, 1979. Rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7: 1513–1523.

    Article  PubMed  CAS  Google Scholar 

  • Bourne, D. G., G. J. Jones, R. L. Blakeley, A. Jones, A. P. Negri & P. Riddles, 1996. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. Applied and Environmental Microbiology 62: 4086–4094.

    PubMed  CAS  Google Scholar 

  • Castenholz, R. W., 2001. Phylum BX. Cyanobacteria. Oxygenic photosynthetic bacteria. In Garrity, G., D. R. Boone & R. W. Castenholz (eds), Bergey’s Manual of Systematic Bacteriology. Volume 1: The Archaea and the Deeply Branching and Phototropic Bacteria, 2nd edition. Springer-Verlag, New York: 473–599.

    Google Scholar 

  • Chernicharo, C. A. L., L. Florencio, R. K. X. Bastos, R. P. Piveli, M. Von Sperling & L. O. Monteggia, 2006. Wastewater treatment and effluent production to adequate different types of water reuse. In Florencio, L., R. K. X. Bastos & M. M. Aisse (eds), PROSAB – Treatment and Utilization of Sanitary Sewage. ABES, Rio de Janeiro: 75–76.

    Google Scholar 

  • CONAMA (Brazilian National Environmental Council), 2005. Resolution 357, Establishes classes of bodies of water, sets forth guidelines for their classification and imposes conditions and standards for the discharge of effluents, among other measures. Ministry of the Environment.

  • Daims, H., M. W. Taylor & M. Wagner, 2006. Wastewater treatment: a model system for microbial ecology. Trends in Biotechnology 24: 483–489.

    Article  PubMed  CAS  Google Scholar 

  • Fiore, M. F., D. H. Moon, S. M. Tsai, H. Lee & J. T. Trevors, 2000. Miniprep DNA isolation from unicellular and filamentous cyanobacteria. Journal of Microbiological Methods 39: 159–169.

    Article  PubMed  CAS  Google Scholar 

  • Furtado, A. L. F. F., 2003. Assessment by denaturing gradient gel electrophoresis (DGGE) of Microcystis (Cyanobacteria) species in a stabilization ponds system in the city of São Lourenço da Serra (Vale do Ribeira de Iguape)-SP. M.Sc. dissertation, São Carlos Engineering School-Hydraulic and Sanitation Department, University of São Paulo: 108 pp.

  • Garcia-Pichel, F., U. Nubel & G. Muyzer, 1998. The phylogeny of unicellular, extremely halotolerant cyanobacteria. Archives of Microbiology 169: 469–482.

    Article  PubMed  CAS  Google Scholar 

  • Gkelis, S., P. Rajaniemi, E. Vardaka, M. Moustaka-Gouni, T. Lanaras & K. Sivonen, 2005. Limnothrix redekei (Van Goor) Meffert (Cyanobacteria) strains from lake Kastoria, Greece form a separated phylogenetic group. Microbial Ecology 49: 176–182.

    Article  PubMed  CAS  Google Scholar 

  • Herdman, M., R. W. Castenholz, I. Iteman & R. Rippka, 2001. Form-genus XIV. Synechocystis. In Garrity, G., D. R. Boone & R. W. Castenholz (eds), Bergey’s Manual of Systematic Bacteriology. Volume 1: The Archaea and the Deeply Branching and Phototropic Bacteria, 2nd edition. Springer-Verlag, New York: 512–514.

    Google Scholar 

  • Izaguirre, G., A. D. Jungblut & B. A. Neilan, 2007. Benthic cyanobacteria (Oscillatoriaceae) that produce microcystin-LR, isolated from four reservoirs in southern California. Water Research 41: 492–498.

    Article  PubMed  CAS  Google Scholar 

  • Jones, G. J., G. Bourne, R. L. Blakeley & H. Doelle, 1994. Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria. Natural Toxins 2: 228–235.

    Article  PubMed  CAS  Google Scholar 

  • Komárek, J. & K. Anagnostidis, 1989. Modern approach to the classification system of cyanophytes, 4 – Nostocales. Algological Studies 56: 247–345.

    Google Scholar 

  • Komárek, J. & K. Anagnostidis, 1999. Cyanoprokaryota, Part 1: Chroococcales, Süsswasserflora von Mitteleuropa, Bd 19/1. Gustav Fischer Verlag, Stuttgart.

  • Komárek, J. & K. Anagnostidis, 2005. Cyanoprokaryota, Part 2: Oscillatoriales, Süsswasserflora von Mitteleuropa, Bd 19/2. Spektrum Akademischer Verlag, Heidelberg.

  • Kumar, S., K. Tamura & M. Nei, 2004. MEGA3, integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5: 150–163.

    Article  PubMed  CAS  Google Scholar 

  • Lane, D. J., 1991. 16S/23S rRNA sequencing. In Stackebrandt, E. & M. Goodfellow (eds), Nucleic Acid Techniques in Bacterial Systematics. Wiley, Chichester: 115–175.

    Google Scholar 

  • Litvaitis, M. K., 2002. A molecular test of cyanobacterial phylogeny: inferences from constraint analyses. Hydrobiologia 468: 135–145.

    Article  CAS  Google Scholar 

  • Marquardt, J. & K. A. Palinska, 2006. Genotypic and phenotypic diversity of cyanobacteria assigned to the genus Phormidium (Oscillatoriales) from different habitats and geographical sites. Archives of Microbiology 187: 397–413.

    Article  PubMed  CAS  Google Scholar 

  • Mohamed, Z. A., M. H. El-Sharouny & W. S. M. Ali, 2006. Microcystin production in benthic mats of cyanobacteria in the Nile River and irrigation canals, Egypt. Toxicon 47: 584–590.

    Article  PubMed  CAS  Google Scholar 

  • Nandini, S., 1999. Variations in physical and chemical parameters and plankton community structure in a series of sewage-stabilization ponds. Revista de Biologia Tropical 47: 149–156.

    Google Scholar 

  • Neilan, B. A., D. Jacobs, T. D. Dot, L. L. Blackall, P. R. Hawkins, P. T. Cox & A. E. Goodman, 1997. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. International Journal of Systematic Bacteriology 47: 693–697.

    Article  PubMed  CAS  Google Scholar 

  • Nelissen, B., R. De Baere, A. Wilmotte & R. De Wachter, 1996. Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequences analysis. Journal of Molecular Evolution 42: 194–200.

    Article  PubMed  CAS  Google Scholar 

  • Oudra, B., M. Loudiki, V. Vasconcelos, B. Sabour, B. Sbiyyaa, K. H. Oufdou & N. Mezrioui, 2002. Detection and quantification of microcystins from cyanobacteria strains isolated from reservoirs and ponds in Morocco. Environmental Toxicology 17: 32–39.

    Article  PubMed  CAS  Google Scholar 

  • Oufdou, K., N. Mezrioul, B. Oudra, M. Barakate, M. Loudiki & A. A. Alla, 2000. Relationship between bacteria and cyanobacteria in the Marrakech waste stabilization ponds. Water Science and Technology 42: 171–178.

    Google Scholar 

  • Palinska, K. A., W. Liesack, E. Rhiel & W. E. Krumbein, 1996. Phenotype variability of identical genotypes: the need for a combined approach in cyanobacterial taxonomy demonstrated on Merismopedia-like isolates. Archives of Microbiology 166: 224–233.

    Article  PubMed  CAS  Google Scholar 

  • Park, H.-D., Y. Sasaki, T. Maruyama, E. Yanagisawa, A. Hiraishi & K. Kato, 2001. Degradation of the cyanobacterial hepatotoxin microcystin by a new bacterium isolated from a hypertrophic lake. Environmental Toxicology 16: 337–343.

    Article  PubMed  CAS  Google Scholar 

  • Pomati, F., S. Sacchi, C. Rosseti, S. Giovannardi, H. Onodera, Y. Oshima & B. A. Neilan, 2000. The freshwater cyanobacterium Planktothrix sp. FP1: molecular identification and detection of paralytic shellfish poisoning toxins. Journal of Phycology 36: 553–562.

    Article  CAS  Google Scholar 

  • Rajaniemi-Wacklin, P., A. Rantala, M. A. Mugnai, S. Turicchia, S. Ventura, J. Komárková, L. Lepistö & K. Sivonen, 2005. Correspondence between phylogeny and morphology of Snowella spp. and Woronichinia naegeliana, cyanobacterial commonly occurring in lakes. Journal of Phycology 42: 226–232.

    Article  Google Scholar 

  • Richardson, L. L., R. Sekar, J. L. Myers, M. Gantar, J. D. Voss, L. Kaczmarsky, E. R. Remily, G. L. Boyer & P. V. Zimba, 2007. The presence of the cyanobacterial toxin microcystin in black band disease of corals. FEMS Microbiology Letters 272: 182–187.

    Article  PubMed  CAS  Google Scholar 

  • Seo, P.-S. & A. Yokota, 2003. The phylogenetic relationships of cyanobacteria inferred from 16S rRNA, gyrB, rpoC1 and rpoD1 gene sequences. Journal of General and Applied Microbiology 49: 191–203.

    Article  PubMed  CAS  Google Scholar 

  • Sivonen, K. & G. Jones, 1999. Cyanobacterial toxins. In Chorus, I. & J. Bartram (eds), Toxic Cyanobacteria in Water. A Guide to Their Public Health Consequences Monitoring and Management. E and FN Spon, London: 41–111.

    Google Scholar 

  • Sivonen, K., W. W. Carmichael, M. Namikoshi, K. L. Rinehart, A. M. Dahlem & S. I. Niemelän, 1990. Isolation and characterization of hepatotoxic microcystin homologues from the filamentous freshwater cyanobacterium Nostoc sp., strain 152. Applied and Environmental Microbiology 56: 2650–2657.

    PubMed  CAS  Google Scholar 

  • Svenning, M. M., T. Eriksson & U. Rasmussen, 2005. Phylogeny of symbiotic cyanobacteria within the genus Nostoc based on 16S rDNA sequence analysis. Archives of Microbiology 183: 19–26.

    Article  PubMed  CAS  Google Scholar 

  • Taton, A., S. Grubisic, D. Ertz, D. A. Hodgson, R. Piccardi, N. Biondi, M. R. Tredici, M. Mainini, D. Losi, F. Martinelli & A. Wilmotte, 2006. Polyphasic study of Antarctic cyanobacterial strains. Journal of Phycology 42: 1257–1270.

    Article  CAS  Google Scholar 

  • Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin & D. G. Higgins, 1997. The CLUSTAL_X windows interface, flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Research 25: 4876–4882.

    Article  CAS  Google Scholar 

  • Tsuji, K., M. Asakawa, Y. Anzai, T. Sumino & K. Harada, 2005. Degradation of microcystins using immobilized microorganism isolated in an eutrophic lake. Chemosphere 65: 117–124.

    Article  CAS  Google Scholar 

  • Urbach, E., D. L. Robertson & S. W. Chisholm, 1992. Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation. Nature 355: 267–270.

    Article  PubMed  CAS  Google Scholar 

  • Urbach, E., D. J. Scanlan, D. L. Distel, J. B. Waterbury & S. W. Chisholm, 1998. Rapid diversification of marine picophytoplankton with dissimilar light-harvesting structures inferred from sequences of Prochlorococcus and Synechococcus (Cyanobacteria). Journal of Molecular Evolution 46: 188–201.

    Article  PubMed  CAS  Google Scholar 

  • Vasconcelos, V. M. & E. Pereira, 2001. Cyanobacteria diversity and toxicity in a wastewater treatment plant (Portugal). Water Research 35: 1354–1357.

    Article  PubMed  CAS  Google Scholar 

  • Von Sperling, M., 1996. Stabilization Ponds. Principles of Biological Treatment of Wastewater, Volume 3. Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

    Google Scholar 

  • Ward, D. M., M. J. Ferris, S. C. Nold & M. M. Bateson, 1998. A natural view of microbial biodiversity within hot spring cyanobacterial mat communities. Microbiology and Molecular Biology Reviews 62: 1353–1370.

    PubMed  CAS  Google Scholar 

  • Willame, R., C. Boutte, S. Grubisic, A. Wilmotte, J. Komárek & L. Hoffmann, 2006. Morphological and molecular characterization of planktonic cyanobacteria from Belgium and Luxembourg. Journal of Phycology 42: 1312–1332.

    Article  CAS  Google Scholar 

  • Wilmotte, A., 1994. Molecular evolution and taxonomy of the cyanobacteria. In Briant, D. A. (ed.), The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht: 1–25.

    Google Scholar 

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Acknowledgments

We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support (2002/13449-1 to M.C.C. and 2005/56303-5 to M.F.F.). A.L.F.F. Furtado and R.Y. Honda received graduate scholarships from CAPES (Ministry of Education Agency) and A.S. Lorenzi received a graduate scholarship from CNPq (National Council for Scientific and Technological Development—140327/2004-5). D.B. Genuário was supported by FAPESP graduate scholarship (2007/06360-8). M.F. Fiore would also like to thank CNPq for a research fellowship (311094/2006-6). We thank Dr. A.C.P. Miwa for the algae and cyanobacteria counting and Dr. L.C.R. Pessenda (Carbon 14 Laboratory, CENA/USP) for permission to use the Axioskop 40 microscope.

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  1. Departamento de Hidráulica e Saneamento, Escola de Engenharia de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense 400, Caixa Postal 359, São Carlos, SP, CEP 13566-590, Brazil

    Ana Luiza Fonseca Fortes Furtado & Maria do Carmo Calijuri

  2. Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário 303, Caixa Postal 96, Piracicaba, SP, CEP 13400-970, Brazil

    Ana Luiza Fonseca Fortes Furtado, Adriana Sturion Lorenzi, Ricardo Yukio Honda, Diego Bonaldo Genuário & Marli Fátima Fiore

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  1. Ana Luiza Fonseca Fortes Furtado
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Furtado, A.L.F.F., Calijuri, M.d.C., Lorenzi, A.S. et al. Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production. Hydrobiologia 627, 195–209 (2009). https://doi.org/10.1007/s10750-009-9728-6

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