Putative glycogen-accumulating organisms belonging to the identified through rRNA-based stable isotope probing Free

Abstract

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of not previously shown to possess the GAO phenotype, but 90 % identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate . Fluorescence hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic–aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6·6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33 % of all in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55 % of ) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified -related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

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2006-02-01
2024-03-29
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References

  1. Altschul S. F, Madden T. L, Zhang J. H, Zhang Z, Miller W, Lipman D. J, Schäffer A. A. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
    [Google Scholar]
  2. Amann R. I. 1995; In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes. In Molecular Microbial Ecology Manual , MMEM–3.3.6/1–MMEM –3.3.6/15 Edited by Akkermans A., van Elsas J., de Bruijn F. London: Kluwer Academic Publications;
    [Google Scholar]
  3. Amann R. I, Binder B. J, Olson R. J, Chisholm S. W, Devereux R, Stahl D.A. 1990; Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
    [Google Scholar]
  4. Beer M, Kong Y. H, Seviour R. J. 2004; Are some putative glycogen accumulating organisms (GAO) in anaerobic : aerobic activated sludge systems members of the Alphaproteobacteria ?. Microbiology 150:2267–2275 [CrossRef]
    [Google Scholar]
  5. Behrens S, Ruhland C, Inacio J, Huber H, Fonseca A, Spencer-Martins I, Fuchs B. M, Amann R. 2003; In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl Environ Microbiol 69:1748–1758 [CrossRef]
    [Google Scholar]
  6. Cech J. S, Hartman P. 1993; Competition between polyphosphate and polysaccharide accumulating bacteria in enhanced biological phosphate removal systems. Water Res 27:1219–1225 [CrossRef]
    [Google Scholar]
  7. Chen Y. G, Chen Y. S, Xu Q, Zhou Q, Gu G. W. 2005; Comparison between acclimated and unacclimated biomass affecting anaerobic–aerobic transformations in the biological removal of phosphorus. Process Biochem 40:723–732 [CrossRef]
    [Google Scholar]
  8. Crocetti G. R, Hugenholtz P, Bond P. L, Schuler A, Keller J, Jenkins D, Blackall L. L. 2000; Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation. Appl Environ Microbiol 66:1175–1182 [CrossRef]
    [Google Scholar]
  9. Crocetti G. R, Banfield J. F, Keller J, Bond P. J, Blackall L. L. 2002; Glycogen accumulating organisms in laboratory-scale and full-scale activated sludge processes. Microbiology 148:3353–3364
    [Google Scholar]
  10. Daims H, Bruhl A, Amann R, Schleifer K. H, Wagner M. 1999; The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444 [CrossRef]
    [Google Scholar]
  11. de Haas D. V. 2005; Nutrient removal in three oxidation ditch plants. AWA Water J Sept56–59
    [Google Scholar]
  12. Erhart R, Bradford D, Seviour R. J, Amann R. I, Blackall L. L. 1997; Development and use of fluorescent in situ hybridization probes for the detection and identification of ‘ Microthrix parvicella ’ in activated sludge. Syst Appl Microbiol 20:310–318 [CrossRef]
    [Google Scholar]
  13. Filipe C. D. M, Daigger G. T, Grady C. P. L. 2001; A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: stoichiometry, kinetics, and the effect of pH. Biotechnol Bioeng 76:17–31 [CrossRef]
    [Google Scholar]
  14. Fuchs B. M, Glockner F. O, Wulf J, Amann R. 2000; Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66:3603–3607 [CrossRef]
    [Google Scholar]
  15. Kong Y, Ong S. L, Ng W. J, Liu W.-T. 2002a; Diversity and distribution of a deeply branched novel proteobacterial group found in anaerobic–aerobic activated sludge processes. Environ Microbiol 4:753–757 [CrossRef]
    [Google Scholar]
  16. Kong Y. H, Beer M, Rees G. N, Seviour R. J. 2002b; Functional analysis of microbial communities in aerobic–anaerobic sequencing batch reactors fed with different phosphorus/carbon (P/C) ratios. Microbiology 148:2299–2307
    [Google Scholar]
  17. Lane D. J. 1992; 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics pp  115–176 Edited by Stackebrandt E., Goodfellow M. London: Wiley;
    [Google Scholar]
  18. Levantesi C, Serafim L. S, Crocetti G. R, Lemos P. C, Rossetti S, Blackall L. L, Reis M. A. M, Tandoi V. 2002; Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor. Environ Microbiol 4:559–569 [CrossRef]
    [Google Scholar]
  19. Liu W. T, Mino T, Nakamura K, Matsuo T. 1996; Glycogen accumulating population and its anaerobic substrate uptake in anaerobic–aerobic activated sludge without biological phosphorus removal. Water Res 30:75–82 [CrossRef]
    [Google Scholar]
  20. Ludwig W, Strunk O, Westram R. other authors 2004; arb: a software environment for sequence data. Nucleic Acids Res 32:1363–1371 [CrossRef]
    [Google Scholar]
  21. Manz W, Amann R, Ludwig W, Wagner M, Schleifer K. H. 1992; Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria – problems and solutions. Syst Appl Microbiol 15:593–600 [CrossRef]
    [Google Scholar]
  22. Maszenan A. M, Seviour R. J, Patel B. K. C, Janssen P. H, Wanner J. 2005; Defluvicoccus vanus gen. nov. sp. nov., a novel Gram-negative coccus/coccobacillus in the Alphaproteobacteria from activated sludge. Int J Syst Evol Microbiol 55:2105–2111 [CrossRef]
    [Google Scholar]
  23. Mino T, Heijnen J. J, van Loosdrecht M. C. M. 1998; Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Res 32:3193–3207 [CrossRef]
    [Google Scholar]
  24. Neef A, Witzenberger R, Kämpfer P. 1999; Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes. J Ind Microbiol Biotechnol 23:261–267 [CrossRef]
    [Google Scholar]
  25. Nielsen A. T, Liu W.-T, Filipe C, Grady L, Molin S, Stahl D. A. 1999; Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Appl Environ Microbiol 65:1251–1258
    [Google Scholar]
  26. Oehmen A, Yuan Z, Blackall L. L, Keller J. 2004; Short-term effects of carbon source on the competition of polyphosphate accumulating organisms and glycogen accumulating organisms. Water Sci Technol 50:139–144
    [Google Scholar]
  27. Oehmen A, Yuan Z, Blackall L. L, Keller J. 2005a; Comparison of acetate and propionate uptake by polyphosphate accumulating organisms and glycogen accumulating organisms. Biotechnol Bioeng 91:162–168 [CrossRef]
    [Google Scholar]
  28. Oehmen A, Zeng R. J, Yuan J, Keller J. 2005b; Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems. Biotechnol Bioeng 91:43–53 [CrossRef]
    [Google Scholar]
  29. Oehmen A, Vives M. T, Lu H, Yuan Z, Keller J. 2005c; The effect of pH on the competition between polyphosphate accumulating organisms and glycogen accumulating organisms. Water Res 39:3727–3737 [CrossRef]
    [Google Scholar]
  30. Ostle A. G, Holt J. G. 1982; Nile blue-a as a fluorescent stain for poly-beta-hydroxybutyrate. Appl Environ Microbiol 44:238–241
    [Google Scholar]
  31. Pijuan M, Saunders A. M, Guisasola A, Baeza J. A, Casas C, Blackall L. L. 2004; Enhanced biological phosphorus removal in a sequencing batch reactor using propionate as the sole carbon source. Biotechnol Bioeng 85:56–67 [CrossRef]
    [Google Scholar]
  32. Sambrook J, Russell D. W. 2001 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  33. Satoh H, Mino T, Matsuo T. 1992; Uptake of organic substrates and accumulation of polyhydroxyalkanoates linked with glycolysis of intracellular carbohydrates under anaerobic conditions in the biological excess phosphate removal processes. Water Sci Technol 26:933–942
    [Google Scholar]
  34. Saunders A. M, Oehmen A, Blackall L. L, Yuan Z, Keller J. 2003; The effect of GAOs on anaerobic carbon requirements in full-scale Australian EBPR plants. Water Sci Technol 47:37–43
    [Google Scholar]
  35. Seviour R. J, Mino T, Onuki M. 2003; The microbiology of biological phosphorus removal in activated sludge systems. FEMS Microbiol Rev 27:99–127 [CrossRef]
    [Google Scholar]
  36. Tchobanoglous G, Burton F. L. 1991 Wastewater Engineering: Treatment, Disposal and Reuse (Metcalf and Eddy), 3rd edn.. New York: McGraw-Hill;
    [Google Scholar]
  37. Tsai C. S, Liu W. T. 2002; Phylogenetic and physiological diversity of tetrad-forming organisms in deteriorated biological phosphorus removal systems. Water Sci Technol 46:179–184
    [Google Scholar]
  38. Wong M. T, Tan F. M, Ng W. J, Liu W. T. 2004; Identification and occurrence of tetrad-forming Alphaproteobacteria in anaerobic–aerobic activated sludge processes. Microbiology 150:3741–3748 [CrossRef]
    [Google Scholar]
  39. Zeng R. J, Lemaire R, Yuan Z, Keller J. 2003a; Simultaneous nitrification, denitrification, and phosphorus removal in a lab-scale sequencing batch reactor. Biotechnol Bioeng 84:170–178 [CrossRef]
    [Google Scholar]
  40. Zeng R. J, Yuan Z. G, Keller J, van Loosdrecht M. C. M. 2003b; Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems. Biotechnol Bioeng 81:92–105 [CrossRef]
    [Google Scholar]
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