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Applied Microbiology and Biotechnology

, Volume 72, Issue 2, pp 393–400 | Cite as

TGGE analysis of the diversity of ammonia-oxidizing and denitrifying bacteria in submerged filter biofilms for the treatment of urban wastewater

  • B. Gómez-Villalba
  • C. Calvo
  • R. Vilchez
  • J. González-LópezEmail author
  • B. Rodelas
Environmental Biotechnology

Abstract

The spatial and temporal diversity of the bacterial community-forming biofilms in a pilot-scale submerged biofilter used for the treatment of urban wastewater was analyzed by a temperature-gradient gel electrophoresis (TGGE) approach. TGGE profiles based on partial sequence of the 16S rRNA gene showed that the community composition of the biofilms remained fairly stable along the column system and during the whole time of operation of the biofilter (more than 1 year). Community-profiling based on the amplification and separation of partial ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes demonstrated that ammonia-oxidizing and denitrifying bacteria coexisted in both the anoxic and the aerated parts of the system. Several amoA and nosZ bands separated by TGGE were reamplified and sequenced, in order to further analyze the composition of these microbial communities in the biofilm. Phylogeny inferred from amoA/AmoA revealed the prevalence of Nitrosomonas species with five sequences affiliated to Nitrosomonas oligotropha, six sequences affiliated to Nitrosomonas europaea, and three sequences that showed only 75.7–76.1% identity of the DNA sequence with the closest described species (Nitrosomonas nitrosa). According to literature, this low identity value is indicative of previously undiscovered species. Eighteen new partial nosZ sequences were obtained which were mostly related to nosZ of gamma-proteobacteria (Pseudomonas) or clustered in the periphery of previously known denitrifying alpha-proteobacteria (Bradyrhizobium and Azospirillum).

Keywords

Chemical Oxygen Demand Ammonia Oxidizer Azospirillum Denitrify Bacterium nosZ Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

This research was supported by Programa Nacional de I+D, Ministerio de Ciencia y Tecnología (MCYT), Spain (AMB99-0666-C02-01). Work by B. Gómez-Villalba was supported by an FPI Ph.D. grant (MCYT, Spain). Work by B. Rodelas was funded by Programa Ramón y Cajal (MCYT, Spain).

References

  1. Altschul SF, Madden TL, Schaeffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedGoogle Scholar
  2. APHA (1995) Standard methods for the examination of water and wastewater, 19th edn. American Public Health Association, Washington DCGoogle Scholar
  3. Bothe H, Günter J, Schloter M, Ward BB, Wizel KP (2000) Molecular analysis of ammonia oxidation and denitrification in natural environments. FEMS Microbiol Rev 24:673–690CrossRefPubMedGoogle Scholar
  4. Calvo C, Gálvez JM, Gómez-Villalba B, Toledo FL, Rodelas B, González-López J (2001) Behaviour of a nitrifying and denitrifying submerged filter in the treatment of urban wastewater. In: Current studies of biotechnology, vol 2. Croatian Society for Biotechnology, p 175–182Google Scholar
  5. Ebie Y, Noda N, Miura H, Matsumura M, Tsuneda S, Hirata A, Inamori Y (2004) Comparative analysis of genetic diversity and expression of amoA in wastewater treatment processes. Appl Microbiol Biotechnol 64:740–744CrossRefPubMedGoogle Scholar
  6. Eichner CA, Erb RW, Timmis KN, Wagner-Dobler I (1999) Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community. Appl Environ Microbiol 65:102–109PubMedGoogle Scholar
  7. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  8. Gálvez JM, Gomez MA, Hontoria E, González-López J (2003) Influence of hydraulic loading and air flowrate on urban wastewater nitrogen removal with a submerged fixed-film reactor. J Hazard Mater 101:219–229CrossRefPubMedGoogle Scholar
  9. Hem LJ, Rusten B, Odegarard H (1994) Nitrification in a moving bed biofilm reactor. Water Sci Technol 28:1425–1433Google Scholar
  10. Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403–405CrossRefPubMedGoogle Scholar
  11. Kimura M (1983) The neutral theory of molecular evolution. Cambridge Univ. Press, Cambridge, U.K., p 75Google Scholar
  12. Lynga A, Balmer P (1992) Denitrification in a non-nitrifying activated sludge system. Water Sci Technol 26:1097–1104Google Scholar
  13. Muyzer G (1999) DGGE/TGGE, a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2:317–322CrossRefPubMedGoogle Scholar
  14. Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73:127–141CrossRefPubMedGoogle Scholar
  15. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16s RNA. Appl Environ Microbiol 59:695–700PubMedGoogle Scholar
  16. Nicolaisen MH, Ramsing NB (2002) Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. J Microbiol Methods 50:189–203CrossRefPubMedGoogle Scholar
  17. Pearson W, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448PubMedCrossRefGoogle Scholar
  18. Pujol R, Hamon H, Kandel X, Lemel H (1994) Biofilters, flexible, reliable biological reactors. Water Sci Technol 29:33–38Google Scholar
  19. Purkhold U, Pommerening-Roser A, Juretschko S, Schmid M, Koops H, Wagner M (2000) Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and AmoA sequence analysis: implications for molecular diversity surveys. Appl Environ Microbiol 66:5386–5382CrossRefGoogle Scholar
  20. Rösch C, Mergel A, Bothe H (2002) Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. Appl Environ Microbiol 68:3818–3829CrossRefPubMedGoogle Scholar
  21. Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  22. Sakano Y, Pickering K, Ström P, Kerkhof LJ (2002) Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support. Appl Environ Microbiol 68:2285–2293CrossRefPubMedGoogle Scholar
  23. Scala DJ, Kerkhof LJ (1998) Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments. FEMS Microbiol Lett 162:61–68CrossRefPubMedGoogle Scholar
  24. Scala DJ, Kerkhof LJ (1999) Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments. Appl Environ Microbiol 65:1681–1687PubMedGoogle Scholar
  25. Throbäck IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:401–417CrossRefGoogle Scholar
  26. Van Loosdrecht MC, Jetten M (1998) Microbiological conversion in nitrogen removal. Water Sci Technol 38:1–7CrossRefGoogle Scholar
  27. Vinuesa P, Rademaker JLW, de Bruijn FJ, Werner D (1998) Genotypic characterization of Bradyrhizobium strains nodulating endemic woody legumes of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16S rRNA (16S rDNA) and 16S-23S rDNA intergenic spacers, repetitive extragenic palindromic PCR genomic fingerprinting, and partial 16S rDNA sequencing. Appl Environ Microbiol 64:2096–2104PubMedGoogle Scholar
  28. Wagner M, Loy AL, Nogueira R, Purkhold U, Lee N, Daims H (2002) Microbial community composition and function in wastewater treatment plants. Antonie van Leeuwenhoek 81:665–680CrossRefPubMedGoogle Scholar
  29. Ward BB, O’Mullan GDO (2002) Worldwide distribution of Nitrosococcus oceani, a marine ammonia-oxidizing γ-proteobacterium, detected by PCR and sequencing of 16S rRNA and amoA genes. Appl Environ Microbiol 68:4153–4157CrossRefPubMedGoogle Scholar
  30. Watanabe K, Yamamoto S, Hino S, Harayama S (1998) Population dynamics of phenol degrading bacteria in activated sludge determined by gyrB-targeted quantitative PCR. Appl Environ Microbiol 64:1203–1209PubMedGoogle Scholar
  31. Watanabe K, Teramoto M, Harayama S (1999) An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process. Appl Environ Microbiol 65:2813–2819PubMedGoogle Scholar
  32. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • B. Gómez-Villalba
    • 1
  • C. Calvo
    • 1
    • 2
  • R. Vilchez
    • 1
  • J. González-López
    • 1
    • 2
    Email author
  • B. Rodelas
    • 1
    • 2
  1. 1.Instituto del AguaUniversidad de GranadaGranadaSpain
  2. 2.Departamento de Microbiología, Facultad de FarmaciaUniversidad de GranadaGranadaSpain

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