Abundance and spatial organization of Gram-negative sulfate-reducing bacteria in activated sludge investigated by in situ probing with specific 16S rRNA targeted oligonucleotides
Werner Manz,
Michael Eisenbrecher,
Thomas R. Neu,
Ulrich Szewzyk,
Corresponding Author
Werner Manz
Technische Universität Berlin, Institut für Technischen Umweltschutz, Fachgebiet Ökologie der Mikroorganismen, Franklinstraße 29, Sekr. OE5, D-10587 Berlin, Germany
*Corresponding author. Tel.: +49 (30) 314 25589; Fax: +49 (30) 314 73461; E-mail: [email protected]Search for more papers by this authorMichael Eisenbrecher
Technische Universität Berlin, Institut für Technischen Umweltschutz, Fachgebiet Ökologie der Mikroorganismen, Franklinstraße 29, Sekr. OE5, D-10587 Berlin, Germany
Search for more papers by this authorThomas R. Neu
UFZ-Umweltforschungszentrum Leipzig-Halle GmbH, Sektion Gewässerforschung, D-39114 Magdeburg, Germany
Search for more papers by this authorUlrich Szewzyk
Technische Universität Berlin, Institut für Technischen Umweltschutz, Fachgebiet Ökologie der Mikroorganismen, Franklinstraße 29, Sekr. OE5, D-10587 Berlin, Germany
Search for more papers by this authorWerner Manz,
Michael Eisenbrecher,
Thomas R. Neu,
Ulrich Szewzyk,
Corresponding Author
Werner Manz
Technische Universität Berlin, Institut für Technischen Umweltschutz, Fachgebiet Ökologie der Mikroorganismen, Franklinstraße 29, Sekr. OE5, D-10587 Berlin, Germany
*Corresponding author. Tel.: +49 (30) 314 25589; Fax: +49 (30) 314 73461; E-mail: [email protected]Search for more papers by this authorMichael Eisenbrecher
Technische Universität Berlin, Institut für Technischen Umweltschutz, Fachgebiet Ökologie der Mikroorganismen, Franklinstraße 29, Sekr. OE5, D-10587 Berlin, Germany
Search for more papers by this authorThomas R. Neu
UFZ-Umweltforschungszentrum Leipzig-Halle GmbH, Sektion Gewässerforschung, D-39114 Magdeburg, Germany
Search for more papers by this authorUlrich Szewzyk
Technische Universität Berlin, Institut für Technischen Umweltschutz, Fachgebiet Ökologie der Mikroorganismen, Franklinstraße 29, Sekr. OE5, D-10587 Berlin, Germany
Search for more papers by this authorAbstract
A comprehensive panel of ten 16S rRNA targeted oligonucleotides specific for mesophilic sulfate-reducing bacteria (SRB) within the δ-subclass of Proteobacteria was developed as a diagnostic tool and evaluated for its specificity and in situ applicability. Five probes (DSD131, DSBO224, DSV407, DSR651, DSS658) are specific on genus level and five probes identify distinct phylogenetic subbranches within the families Desulfobacteriaceae (DSMA488, DSB985) and Desulfovibrionaceae (DSV214, DSV698, DSV1292). All oligonucleotides were checked for their specificity by computer aided comparative sequence analysis. For in situ application optimal stringency conditions were adjusted for each fluorescence-labelled probe performing whole cell hybridizations using target and non-target organisms. Activated sludge flocs from different stages within the aeration tank of a large municipal wastewater treatment plant were examined by in situ hybridizations with the indocarbocyanine- (Cy3-) labelled SRB-specific probes. The relative abundance and the spatial organization of single SRB were monitored with epifluorescence and confocal laser scanning microscopy. Individual sulfate-reducing cells could be visualized and the number of cells ranged from 0.5 to 8% of the total cell counts within all stages of the activated sludge process and the final clarifier. Cells yielding strong fluorescence signals after hybridization with the newly developed probes were not restricted to anoxic and anaerobic compartments, but were also clearly detectable in the aeration zones of the treatment plant.
References
- 1 Fowler, V.J., Widdel, F., Pfennig, N., Woese, C.R. and Stackebrandt, E. (1986) Phylogenetic relationships of sulfate- and sulfur-reducing bacteria. Syst. Appl. Microbiol. 8, 32–41.
- 2 Devereux, R., He, S.H., Doyle, C.L., Oakland, S., Stahl, D.A., LeGall, J. and Whitman, W.B. (1990) Diversity and origin of Desulfovibrio species: phylogenetic definition of a family. J. Bacteriol. 172, 3609–3619.
- 3 Devereux, R., Kane, M.D., Winfrey, J. and Stahl, D.A. (1992) Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria. Syst. Appl. Microbiol. 15, 601–609.
- 4 Devereux, R., Delaney, M., Widdel, F. and Stahl, D.A. (1989) Natural relationships among sulfate-reducing eubacteria. J. Bacteriol. 171, 6689–6695.
- 5 Muyzer, G. and Ramsing, N.B. (1995) Molecular methods to study the organization of microbial communities. Water Sci. Technol. 32, 1–9.
- 6 Amann, R.I., Ludwig, W. and Schleifer, K.-H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169.
- 7 Jannasch, H.W. and Jones, G.E. (1959) Bacterial populations in seawater as determined by different methods of enumeration. Limnol. Oceanogr. 4, 128–139.
- 8 Staley, J.T. and Konopka, A. (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39, 321–346.
- 9 Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R. and 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.
- 10 Rabus, R., Fukui, M., Wilkes, H. and Widdel, F. (1996) Degradative capacities and 16S rRNA-targeted whole cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil. Appl. Environ. Microbiol. 62, 3605–3613.
- 11 Amann, R.I., Stromley, J., Devereux, R., Key, R. and Stahl, D.A. (1992) Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl. Environ. Microbiol. 58, 614–623.
- 12 Kane, M.D., Poulsen, L.K. and Stahl, D.A. (1993) Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences. Appl. Environ. Microbiol. 59, 682–686.
- 13 Poulsen, L.K., Ballard, G. and Stahl, D.A. (1993) Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms. Appl. Environ. Microbiol. 59, 1354–1360.
- 14 Ramsing, N.B., Kühl, M. and Jørgensen, B.B. (1993) Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes. Appl. Environ. Microbiol. 59, 3840–384.
- 15 Devereux, R. and Mundfrom, G.W. (1994) A phylogenetic tree of 16S rRNA sequences from sulfate-reducing bacteria in a sandy marine sediment. Appl. Environ. Microbiol. 60, 3437–3439.
- 16 Risatti, J.B., Capman, W.C. and Stahl, D.A. (1994) Community structure of a microbial mat: the phylogenetic dimension. Proc. Natl. Acad. Sci. USA 91, 10173–10177.
- 17 Abram, J.W. and Nedwell, D.B. (1978) Inhibition of methanogenesis by sulfate-reducing bacteria competing for transferred hydrogen. Arch. Microbiol. 117, 89–92.
- 18 Winfrey, M.R. and Zeikus, J.G. (1977) Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments. Appl. Environ. Microbiol. 33, 275–281.
- 19 Winfrey, M.R. and Ward, D.M. (1983) Substrates for sulfate reduction and methane production in intertidal sediments. Appl. Environ. Microbiol. 45, 193–199.
- 20 Bak, F. and Pfennig, N. (1991) Sulfate-reducing bacteria in littoral sediment of Lake Constance. FEMS Microbiol. Ecol. 85, 43–52.
- 21 Smith, R.L. and Klug, M.L. (1981) Reduction of sulfur compounds in the sediments of a eutrophic lake basin. Appl. Environ. Microbiol. 41, 1230–1237.
- 22 Canfield, D.E. and DesMarais, D.J. (1991) Aerobic sulfate reduction in microbial mats. Science 251, 1471–1473.
- 23 Fründ, C. and Cohen, Y. (1992) Diurnal cycles of sulfate reduction under oxic conditions in cyanobacterial mats. Appl. Environ. Microbiol. 58, 70–77.
- 24 Abdollahi, H. and Wimpenny, J.W.T. (1990) Effects of oxygen on the growth of Desulfovibrio desulfuricans. J. Gen. Microbiol. 136, 1025–1030.
- 25 Dilling, W. and Cypionka, H. (1990) Aerobic respiration in sulfate-reducing bacteria. FEMS Microbiol. Lett. 71, 123–128.
- 26 Van Niel, E.W.J., Pedro Gomes, T.M., Willems, A., Collins, M.D., Prins, R.A. and Gottschal, J.C. (1996) The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens. FEMS Microbiol. Ecol. 21, 243–253.
- 27 Cypionka, H., Widdel, F. and Pfennig, N. (1985) Survival of sulfate-reducing bacteria after oxygen stress, and growth in sulfate-free oxygen-sulfide gradients. FEMS Microbiol. Ecol. 31, 39–45.
- 28 Fukui, M. and Takii, S. (1990) Survival of sulfate-reducing bacteria in oxic surface sediment of a seawater lake. FEMS Microbiol. Ecol. 73, 317–322.
- 29
Marschall, C., Frenzel, P. and Cypionka, H. (1993) Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria.
Arch. Microbiol.
159, 168–173.
10.1007/BF00250278 Google Scholar
- 30 Jørgensen, B.B. and Bak, F. (1991) Pathways and microbiology of thiosulfate transformations and sulfate reduction in a marine sediment (Kattegat, Denmark). Appl. Environ. Microbiol. 57, 847–856.
- 31 Sass, H., Cypionka, H. and Babenzien, H.-D. (1997) Vertical distribution of sulfate-reducing bacteria at the oxic-anoxic interface in sediments of the oligotrophic lake stechlin. FEMS Microbiol. Ecol. 22, 245–255.
- 32 Widdel, F. (1988) Microbiology and ecology of sulfate- and sulfur-reducing bacteria. In: Biology of Anaerobic Microorganisms (Zehnder, A.J.B., Ed.), pp. 469–585. Wiley and Sons, New York.
- 33 Kühl, M. and Jørgensen, B.B. (1992) Microsensor measurements of sulfate reduction and sulfide oxidation in compact microbial communities of aerobic biofilms. Appl. Environ. Microbiol. 58, 1164–1174.
- 34 Lens, P.N., De Poorter, M.-P., Cronenberg, C.C. and Verstraete, W.H. (1995) Sulfate reducing and methane producing bacteria in aerobic wastewater treatment systems. Water Res. 29, 871–880.
- 35 Southwick, P.L., Ernst, L.A., Tauriello, E.W., Parker, S.R., Mujumdar, R.B., Mujumdar, S.R., Clever, H.A. and Waggoner, A.S. (1990) Cyanine dye labeling reagents - carboxymethylindolecyanine succinimidyl esters. Cytometry 11, 418–430.
- 36 American Type Culture Collection (1989) Catalogue of Bacteria and Bacteriophages, 17th Edn., Rockville, MD.
- 37 German Collection of Microorganisms and Cell Cultures (1993) Catalogue of Strains, 5th Edn., Braunschweig.
- 38 Manz, W., Wagner, M., Amann, R. and Schleifer, K.-H. (1994) In situ characterization of the microbial consortia active in two wastewater treatment plants. Water Res. 28, 1715–1723.
- 39 Ludwig, W. (1995) Sequence databases. In: Molecular Microbial Ecology Manual (Akkermans, A.D.L., van Elsas, J.D. and de Bruijn, F.J., Eds.), ch. 3.3.5, pp. 1–22. Kluwer Academic Publishers, Dordrecht.
- 40 Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
- 41 Wessendorf, M.W. and Brelje, T.C. (1992) Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18. Histochemistry 98, 81–85.
- 42 Manz, W., Amann, R.I., Ludwig, W., Wagner, M. and Schleifer, K.-H. (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15, 593–600.
- 43 Stackebrandt, E., Stahl, D.A. and Devereux, R. (1995) Taxonomic relationships. In: Sulfate-Reducing Bacteria (Barton, L.L., Ed.), pp. 49–87. Plenum Press, New York.
- 44
Wallrabenstein, C., Gorny, N., Springer, N., Ludwig, W. and Schink, B. (1995) Pure culture of Syntrophus buswellii, definition of its phylogenetic status, and description of Syntrophus gentianae sp.
nov. Syst. Appl. Microbiol.
18, 62–66.
10.1016/S0723-2020(11)80449-2 Google Scholar
- 45 Ramsing, N.B., Fossing, H., Ferdelman, T.G., Andersen, F. and Thamdrup, B. (1996) Distribution of bacterial populations in a stratified fjord (Mariager fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column. Appl. Environ. Microbiol. 62, 1391–1404.
- 46 Amann, R. (1995) Fluorescently labelled, rRNA-targeted oligonucleotide probes in the study of microbial ecology. Mol. Ecol. 4, 543–554.
- 47 Stahl, D.A. (1995) Application of phylogenetically based hybridization probes to microbial ecology. Mol. Ecol. 4, 535–542.
- 48 Stackebrandt, E., Wehmeyer, U. and Schink, B. (1989) The phylogenetic status of Pelobacter acidigallici; Pelobacter venetianus, and Pelobacter carbinolicus. Syst. Appl. Microbiol. 11, 257–260.
- 49 Lovley, D.R., Giovannoni, S.J., White, D.C., Champine, J.E., Phillips, J.P., Gorby, Y.A. and Goodwin, S. (1993) Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals. Arch. Microbiol. 159, 336–344.
- 50 Widdel, F. and Bak, F. (1992) Gram-negative mesophilic sulfate-reducing bacteria. In: The Prokaryotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications (Balows, A., Trüper, H.G., Dworkin, M., Harder, W. and Schleifer, K.-H., Eds.), 2nd Edn., pp. 3352–3378. Springer-Verlag, New York.
- 51 Devereux, R. and Stahl, D.A. (1993) Phylogeny of sulfate-reducing bacteria and a perspective for analyzing their natural communities. In: The Sulfate-Reducing Bacteria: Contemporary Perspectives (Odom, J.M. and Singleton Jr., R., Eds.), pp. 131–160. Springer-Verlag, New York.
- 52 Lonergan, D.J., Jenter, H.L., Coates, J.D., Phillips, E.J.P., Schmidt, T.M. and Lovely, D.R. (1996) Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria. J. Bacteriol. 178, 2402–2408.
- 53 Flärdh, K., Cohen, P.S. and Kjelleberg, S. (1992) Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956. J. Bacteriol. 174, 6780–6788.
- 54 Kemp, P.F., Lee, S. and LaRoche, J. (1993) Estimating the growth rate of slowly growing marine bacteria from RNA content. Appl. Environ. Microbiol. 59, 2594–2601.
- 55 Schaechter, M., Maaloe, O. and Kjeldgaard, N.O. (1958) Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 19, 592–606.
- 56 Wagner, M., Amann, R., Lemmer, H. and Schleifer, K.-H. (1993) Probing activated sludge with proteobacteria-specific oligonucleotides: inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59, 1520–1525.
- 57 Wanner, J., Kucman, K., Ottova, V. and Grau, P. (1987) Effect of anaerobic conditions on activated sludge filamentous bulking in laboratory systems. Water Res. 21, 1541–1546.
- 58 Yamamoto, R.I., Komori, T. and Matsui, S. (1991) Filamentous bulking and hindrance of phosphate removal due to sulfate reduction in activated sludge. Water Sci. Technol. 23, 927–935.
- 59 Buisman, C.J., Stams, A.J., Meijer, H. and Lettinga, G. (1989) Sulfur and sulfate reduction with acetate and propionate in an aerobic process for sulfide removal. Appl. Microbiol. Biotechnol. 32, 363–370.
- 60 Takahashi, M. and Kyosai, S. (1991) Pilot plant study on microaerobic self-granulated sludge process (multi-stage reversing flow bioreactor: MRB). Water Sci. Technol. 23, 973–980.
- 61 Zietz, U. (1995) The formation of sludge bulking in the activated sludge process. Eur. Water Pollut. Contr. 5, 21–27.
- 62 Oremland, R.S. and Capone, D.G. (1988) Use of ‘specific’ inhibitors in biogeochemistry and microbial ecology. In: Advances in Microbial Ecology (Marshall, K.C., Ed.), pp. 285–383. Plenum Press, New York.
- 63 Hamilton, W.A. (1985) Sulphate-reducing bacteria and anaerobic corrosion. Annu. Rev. Microbiol. 39, 195–217.
- 64 Brosius, J., Dull., T.L., Sleeter, D.D. and Noller, H.F. (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148, 107–127.
