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Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

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Abstract

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.

As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera.

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References

  1. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925

    Google Scholar 

  2. Amann RI, Krumholz L, Stahl DA (1990) Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bact 172:762–770

    Google Scholar 

  3. Amann RI, Springer N, Ludwig W, Görtz H-D, Schleifer K-H (1991) Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351:161–164

    Google Scholar 

  4. Amann RI, Stromley J, Devereux R, Key R, Stahl DA (1992) Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl Environ Microbiol 58: 614–623

    Google Scholar 

  5. Arvin E (1979) The influence of pH and calcium ions upon phosphorus transformations in biological wastewater treatment plants. Prog Water Technol (Suppl) 1:19–40

    Google Scholar 

  6. Arvin E (1985) Biological removal of phosphorus from wastewaters. CRC Crit Rev Environ Control 15:25–64

    Google Scholar 

  7. Bark K, Sponner A, Kämpfer P, Grund S, Dott W (1992) Differences in polyphosphate accumulation and phosphate adsorption by Acinetobacter isolates from wastewater producing polyphosphate: AMP phosphotransferase. Water Res 26:1379–1388

    Google Scholar 

  8. Bark K, Kämpfer P, Sponner A, Dott W (1993) Polyphosphate-dependent enzymes in some coryneform bacteria isolated from sewage sludge. FEMS Microbiol Lett 107:133–138

    Google Scholar 

  9. Bayly RC, Duncan A, May JW, Schembri M, Semertjis A, Vasiliadis G, Raper WGC (1991) Microbiological and genetic aspects of the synthesis of polyphosphate by species of Acinetobacter. Water Sci Technol 23:747–757

    Google Scholar 

  10. Bianchi MAG, Bianchi AIM (1982) Statistical sampling of bacterial strains and its use in bacterial diversity measurements. Microb Ecol 8:61–69

    Google Scholar 

  11. Bohlool BB, Schmidt EL (1980) The immunofluorescence approach in microbial ecology. In: Alexander M (ed) Advances in microbial ecology, vol 4. Plenum Press, New York, pp 203–241

    Google Scholar 

  12. Brock TD (1987) The study of microorganisms in situ: progress and problems. In: Ecology of microbial communities. (Symposium 41) Society for General Microbiology, Cambridge University Press, Cambridge, pp 1–17

    Google Scholar 

  13. Buchan L (1983) Possible biological mechanism of phosphorus removal. Water Sci Technol 15:87–103

    Google Scholar 

  14. Collins MD, Martinez-Murcia AJ, Cai J (1993) Aeromonas enteropelogenes and Aeromonas ichthiosmia are identical to Aeromonas trota and Aeromonas veronii, respectively, as revealed by small-subunit rRNA sequence analysis. Int J Syst Bacteriol 43:855–856

    Google Scholar 

  15. Deinema MH, Habets LHA, Scholten J, Turkstra E, Webers HAAM (1980) The accumulation of polyphosphate in Acinetobacter spp. FEMS Microb Lett 9:275–279

    Google Scholar 

  16. Deinema MH, van Loosdrecht M, Scholten A (1985) Some physiological characteristics of Acinetobacter spp. accumulating large amounts of phosphate. Water Sci Technol 17:119–125

    Google Scholar 

  17. DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single microbial cells. Science 243:1360–1363

    Google Scholar 

  18. De Vos P, De Ley J (1983) Intra- and intergeneric similarities of Pseudomonas and Xanthomonas ribosomal ribonucleic acid cistrons. Int J Syst Bacteriol 33:487–509

    Google Scholar 

  19. Doetsch RN (1981) Determinative methods in light microscopy. In: Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips EB (eds) Manual of methods for general microbiology. American Society for Microbiology, Washington DC, pp 21–33

    Google Scholar 

  20. Duncan A, Vasiliadis GE, Bayly RC, May JW (1988) Genospecies of Acinetobacter isolated from activated sludge showing enhanced removal of phosphate during pilot-scale treatment of sewage. Biotechnol Lett 10:831–836

    Google Scholar 

  21. Eikelboom DH (1975) Filamentous organisms observed in activated sludge. Water Res 9:365–388

    Google Scholar 

  22. Eikelboom DH, van Buijsen HJJ (1983) Microscopic sludge investigation manual. (IMG-TNO Report Nr. A 94) TNO Delft, The Netherlands

    Google Scholar 

  23. Fuhs GW, Chen M (1975) Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microb Ecol 2:119–138

    Google Scholar 

  24. Hahn D, Amann RI, Ludwig W, Akkermans ADL, Schleifer K-H (1992) Detection of microorganisms in soil after in situ hybridization with rRNA-targeted, fluorescently labeled oligonucleotides. J Gen Microbiol 138:879–887

    Google Scholar 

  25. Hao OJ, Chang CH (1987) Kinetics of growth and phosphate uptake in pure culture studies of Acinetobacter species. Biotech Bioeng 29:819–831

    Google Scholar 

  26. Hicks R, Amann RI, Stahl DA (1992) Dual staining of natural bacterioplankton with 4′,6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences. Appl Environ Microbiol 58:2158–2163

    Google Scholar 

  27. Howgrave-Graham AR, Steyn PL (1988) Application of the fluorescent-antibody technique for the detection of Sphaerotilus natans in activated sludge. Appl Environ Microbiol 54:799–802

    Google Scholar 

  28. Jenkins D, Richard MG, Daigger GT (1993) Manual on the causes and control of activated sludge bulking and foaming. Lewis Publ. New York

    Google Scholar 

  29. Kämpfer P, Don W (1989) Numerische Identifizierung aquatischer Mikroorganismen mittels automatisierter Methoden am Beispiel von Bakterien aus dem belebten Schlamm. Zbl Bakt Hyg B 187:216–229

    Google Scholar 

  30. Kämpfer P, Altwegg M (1992) Numerical classification and identification of Aeromonas genospecies. J Appl Bacteriol 72:341–351

    Google Scholar 

  31. Kämpfer P, Eisenträger A, Hergt V, Dott W (1990) Untersuchungen zur bakteriellen Phosphateliminierung. I. Mitteilung: Bakterienflora und bakterielles Phosphatspeicherungsvermögen in Abwasserreinigungsanlagen. gwf/ wasser-abwasser 131:156–164

    Google Scholar 

  32. Kämpfer P, Steiof M, Dort W (1991) Microbiological characterization of a fuel oil contaminated site including numerical identification of heterotrophic water and soil bacteria. Microb Ecol 21:227–251

    Google Scholar 

  33. Kämpfer P, Bark K, Busse H-J, Auling G, Dott W (1992) Numerical and chemotaxonomy of polyphosphate accumulating Acinetobacter strains with high polyphosphate:AMP phosphotransferase (PPAT) activity. System Appl Microbiol 15:409–419

    Google Scholar 

  34. Kämpfer P, Tjernberg I, Ursing J (1993) Numerical classification and identification of Acinetobacter genomic species. J Appl Bacteriol 75:259–268

    Google Scholar 

  35. Larsen N, Olsen GJ, Maidak BL, McCaughey MJ, Overbeek R, Macke TJ, Marsh TL, Woese CR (1993) The ribosomal database project. Nucleic Acids Res 21:3021–3023

    Google Scholar 

  36. Lemmer H (1986) The ecology of scum causing actinomycetes in sewage treatment plants. Water Res 20:531–535

    Google Scholar 

  37. Lemmer H, Baumann M (1988) Scum actinomycetes in sewage treatment plants. Part 3. Synergisms with other sludge bacteria. Water Res 22:765–767

    Google Scholar 

  38. Manz W, Amann R, Ludwig W, Wagner M, Schleifer K-H (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. System Appl Microbiol 15:593–600

    Google Scholar 

  39. Manz W, Amann R, Ludwig W Vancanneyt M, Schleifer K-H (1995) Whole cell hybridization probes for members of the cytophaga-flavobacterium phylum. System Appl Microbiol (in press)

  40. Martinez-Murcia AJ, Esteve C, Garay E, Collins MD (1992) Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas. FEMS Microbiol Lett 91:199–206

    Google Scholar 

  41. Martinez-Murcia AJ, Benlloch S, Collins MD (1992) Phylogenetic interrrelationship of members of the genera Aeromonas and Plesiomonas as determined by 16S rRNA sequencing—lack of congruence with results of DNA-DNA hybridizations. Int J Syst Bacteriol 42:412–421

    Google Scholar 

  42. Mino T, Kawakami T, Matsudo T (1985) Location of phosphorus in activated sludge and function of intracellular polyphosphates in biological phosphorus removal process. Water Sci Technol 17:93–106

    Google Scholar 

  43. Mino T, Arun V, Tsusuki Y, Matsuo T (1987) Effect of phosphorus accumulation on acetate metabolism in the biological phosphate removal process. In: Ramadori R (ed) Biological phosphate removal from wastewater. Pergamon Press, Oxford, pp 27–38

    Google Scholar 

  44. Nakamura K, Masuda K, Mikami E (1989) Polyphosphate accumulating bacteria and their ecological characteristics in activated sludge process. In: Hattori T, Ishida Y, Maruyama Y, Morita R, Uchida A (eds) Recent advances in microbial ecology. Japan Scientific Societies Press, Toyko

    Google Scholar 

  45. Neefs JM, Vandepeer Y, DeRijk P, Chapelle S, De Wachter R (1993) Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res 21:3025–3049

    Google Scholar 

  46. Ohsumi T, Shoda M, Udaka S (1980) Influence of cultural conditions on phosphate accumulation of Arthrobacter globiformis PAB-6. Agric Biol Chem 44:325–331

    Google Scholar 

  47. Othake H, Takahashi K, Tsuzuki Y, Toda K (1985) Uptake and release of phosphate by a pure culture of Acinetobacter calcoaceticus. Water Res 19:1587–1594

    Google Scholar 

  48. Paepcke BH (1983) Performance and operational aspects of biological phosphate removal plants in South Africa. Water Sci Technol 15:219–232

    Google Scholar 

  49. Pfennig N, Lippert KD (1966) Über das Vitamin B12 Bedürfnis phototropher Schwefelbakterien. Arch Microbiol 55:245–256

    Google Scholar 

  50. Porter KG, Feig YS (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr 25:943–948

    Google Scholar 

  51. Prakasam TBS, Dondero NC (1967) Aerobic heterotrophic populations of sewage and activated sludge. I. Enumeration. Appl Microbiol 15:461–467

    Google Scholar 

  52. Reasoner DJ, Geldreich EE (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49:1–7

    Google Scholar 

  53. Rees GN, Vasiliadis G, May JW, Bayly RC (1992) Differentiation of polyphosphate and poly-ß-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge. FEMS Microbiol Lett 94:171–174

    Google Scholar 

  54. Reichenbach H (1992) The order Cytophagales. In: Balows A, Trüper H-G, Dworkin M, Harder W, Schleifer K-H (eds) The prokaryotes. Springer-Verlag, New York, pp 3631–3675.

    Google Scholar 

  55. Rensink JH, Donker HJGW (1984) Biologische Phosphorelimination aus Abwasser. Gwf/Wasser Abwasser 125:238–245

    Google Scholar 

  56. Roller C, Wagner M, Amann R, Ludwig W, Schleifer K-H (1994) In situ probing of Gram-positive bacteria with a high DNA G+C content. Microbiology 140:2849–2858

    Google Scholar 

  57. Ruimy R, Breittmayer V, Elbaze P, Lafay P, Boussemart O, Gauthier M, Christen R (1994) Phylogenetic analysis of the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas deduced from small-subunit rRNA sequences. Int J Syst Bacteriol 44:416–426

    Google Scholar 

  58. Segers P, Vancanneyt M, Pot B, Torck U, Hoste W, Dewettinck D, Falsen E, Kersters K, DeVos P (1994) Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Büsing, Döll, and Freitag 1953 in Brevundimonas gen. nov. as Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively. Int J Syst Bacteriol 44: 499–510

    Google Scholar 

  59. Sneath PHA (1979) BASIC program for identification of an unknown with presence-absence data against an identification matrix of percent positive characters. Comput Geosci 5:195–213

    Google Scholar 

  60. Soddell JA, Seviour RJ (1990) Microbiology of foaming in activated sludge plants. J Appl Bacteriol 69:145–176

    Google Scholar 

  61. Soddell JA, Beacham AM, Seviour RJ (1993) Phenotypic identification of nonclinical isolates of Acinetobacter species. J Appl Bacteriol 74:210–214

    Google Scholar 

  62. Spring S, Amann R, Ludwig W, Schleifer K-H, Petersen N (1992) Phylogenetic diversity and identification of nonculturable magnetotactic bacteria. System Appl Microbiol 15:116–122

    Google Scholar 

  63. Stahl DA, Amann R (1991) Development and application of nucleic acid probes in bacterial systematics. In: Stackebrandt E, Goodfellow M (eds) Sequencing and hybridization techniques in bacterial systematics. John Wiley and Sons, Chichester, England pp 205–248

    Google Scholar 

  64. Stahl DA, Flesher B, Mansfield HR, Montgomery L (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol 54: 1079–1084

    Google Scholar 

  65. Stahl DA, Devereux R, Amann RI, Flesher B, Stromley J (1989) Ribosomal RNA based studies of natural microbial diversity and ecology. In: Hattori T, Ishida Y, Maruyama Y, Morita R, Uchida A (eds), Recent advances in microbial ecology. Japan Scientific Societies Press, Tokyo, Japan, pp 669–673

    Google Scholar 

  66. Streichan M, Golecki JR, Schön G (1990) Polyphosphate-accumulating bacteria from sewage plants with different processes for biological phosphorus removal. FEMS Microb Ecol 73:113–124

    Google Scholar 

  67. van Groenestijn JW, Deinema M, Zehnder AJB (1987) ATP production from polyphosphate in Acinetobacter strain 210A. Arch Microbiol 148:14–19

    Google Scholar 

  68. Vasiliadis G, Duncan A, Bayly RC, May JW (1990) Polyphosphate production by strains of Acinetobacter. FEMS Microbiol Lett 70:37–40.

    Google Scholar 

  69. Wagner M, Amann R, Lemmer H, Schleifer K-H (1993) Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. Appl Environ Microbiol 59:1520–1525

    Google Scholar 

  70. Wagner M, Amann R, Kämpfer P, Aßmus B, Hartmann A, Hutzler P, Springer N, Schleifer K-H (1994) Identification and in situ detection of Gram-negative filamentous bacteria in activated sludge. System Appl Microbiol 17:405–417

    Google Scholar 

  71. Wagner M, Erhart R, Manz W, Amann R, Lemmer H, Wedi D, Schleifer K-H (1994) Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring of activated sludge. Appl Environ Microbiol 60:792–800

    Google Scholar 

  72. Wedi D, Wilderer PA (1993) Full scale investigations on enhanced biological phosphorus removal-P-release in the anaerobic reactor. IAWQ international specialized conference on microorganisms in activated sludge and biofilm processes, Paris, 27–29 September, 1993

  73. Wentzel MC, Lötter LH, Loewenthal RE, Marais GvR (1986) Metabolic behaviour of Acinetobacter spp. in enhanced biological phosphate removal. Water SA 12:209–223

    Google Scholar 

  74. Wentzel MC, Lötter LH, Ekema GA, Loewenthal RE, Marais GvR (1991) Evaluation of biochemical models for biological excess phosphorus removal. Water Sci Technol 23:567–576

    Google Scholar 

  75. Willcox WR, Lapage SP, Bascomb S, Curtis MA (1973) Identification of bacteria by computer: theory and progamming. J Gen Microbiol 77:317–330

    Google Scholar 

  76. Zarda B, Amann R, Wallner G, Schleifer K-H (1991) Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. J Gen Microbiol 137:2823–2830

    Google Scholar 

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  1. P. Kämpfer

    Present address: Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Senckenbergstr. 3, D-35390, Giessen, Germany

Authors and Affiliations

  1. Fachgebiet Hygiene, Technische Universität Berlin, Amrumerstr. 32, D-13353, Berlin, Germany

    P. Kämpfer, J. Böhringer & M. Wagner

  2. Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Senckenbergstr. 3, D-35390, Giessen, Germany

    P. Kämpfer

  3. Lehrstuhl für Mikrobiologie, Technische Universität München, Arcisstr, 21, D-80290, München, Germany

    R. Erhart, C. Beimfohr & R. Amann

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Kämpfer, P., Erhart, R., Beimfohr, C. et al. Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes. Microb Ecol 32, 101–121 (1996). https://doi.org/10.1007/BF00185883

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