Applied Microbiology and Biotechnology

, Volume 63, Issue 3, pp 322–334 | Cite as

Sulfate-reducing bacterial community structure and their contribution to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions

  • S. OkabeEmail author
  • T. Ito
  • H. Satoh
Original Paper


The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga-Flexibacter-Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H2 as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2–4×109 cells cm–3 and 5×107 filaments cm–3, respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB.


Sulfate Reduction Desulfovibrio Acid Volatile Sulfide Sulfate Reduction Rate Microaerophilic Condition 
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.



This study was carried out as part of "The Project for Development of Technologies for Analyzing and Controlling the Mechanism of Biodegrading and Processing", which was initiated by the New Energy and Industrial Technology Development Organization (NEDO). This research was also partly supported by Grant-in Aid (No.13650593) for Developmental Scientific Research from the Ministry of Education, Science and Culture of Japan.


  1. Achenbach LA, Michaelidou U, Bruce RA, Fryman J, Coates JD (2001) Dechloromonas agitata gen., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position. Int J Syst Evol Microbiol 51:527–533PubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer 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 25:3389–3402PubMedGoogle Scholar
  3. Amann RI (1995) In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes. In: Akkerman ADL, van Elsas JD, de Bruijn FJ (eds) Molecular microbial ecology manual. Kluwer, Dordrecht, pp 1–15Google Scholar
  4. Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770PubMedGoogle Scholar
  5. Amann RI, Stomley J, Devereux RK, Stahl DA (1992) Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl Environ Microbiol 58:614–623PubMedGoogle Scholar
  6. Andrussow L (1969) Diffusion. In: Landolt-Börnstein Zahlenw Funkt, vol II5a, 6th edn. Springer, Berlin Heidelberg New York, pp 513–727Google Scholar
  7. Beer D de, Schramm A, Santegoeds CM, Kühl M (1997) A nitrite microsensor for profiling environmental biofilms. Appl Environ Microbiol 63:973–977Google Scholar
  8. Bond PL, Hugenholtz P, Keller J, Blackall LL (1995) Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Appl Environ Microbiol 61:1910–1916PubMedGoogle Scholar
  9. Canfield DE, Des Marais DJ (1991) Aerobic sulfate reduction in microbial mats. Science 251:1471–1473PubMedGoogle Scholar
  10. Cline JD (1969) Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr 14:454–458Google Scholar
  11. Devereux R, Kane MD, Winfrey J, Stahl DA (1992) Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria. Syst Appl Microbiol 15:601–609Google Scholar
  12. Fukui M, Teske A, Assmus B, Muyzer G, Widdel F (1999) Physiology, phylogenetic relationships, and ecology of filamentous sulfate-reducing bacteria (genus Desulfonema). Arch Microbiol 172:193–203CrossRefPubMedGoogle Scholar
  13. Godon J-J, Zumstein E, Dabert P, Habouzit F, Moletta R (1997) Molecular microbial diversity of an anaerobic digester as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 63:2802–2813PubMedGoogle Scholar
  14. Hesselmann RPX, Werlen C, Hahn D, van der Meer JR, Zehnder AJB (1999) Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Syst Appl Microbiol 22:454–465PubMedGoogle Scholar
  15. Ito T, Nielsen JL, Okabe S, Watanabe Y, Nielsen P H (2002a) Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm by combining microautoradiography and fluorescent in situ hybridization. Appl Environ Microbiol 68:356–364CrossRefPubMedGoogle Scholar
  16. Ito T, Okabe S, Satoh H, Watanabe Y (2002b) Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions. Appl Environ Microbiol 68:1392–1402CrossRefPubMedGoogle Scholar
  17. Kühl M, Jørgensen BB (1992) Microsensor measurement of sulfate reduction and sulfide oxidation in compact microbial communities of aerobic biofilms. Appl Environ Microbiol 58:1164–1174Google Scholar
  18. Lens PN, Sipma J, Hulshoff Pul LW, Lettinga G (2000) Effect of nitrate on acetate degradation in a sulfidogenic staged reactor. Water Res 34:31–42CrossRefGoogle Scholar
  19. Liu W-T, Chan O-C, Fang HHP (2002) Characterization of microbial community in granular sludge treating brewery wastewater. Water Res 36:1767–1775CrossRefPubMedGoogle Scholar
  20. Lorenzen J, Larsen LH, Kjar T, Revsbech NP (1998) Biosensor detection of the microscale distribution of nitrate, nitrate assimilation, nitrification, and denitrification in a diatom-inhabited freshwater sediment. Appl Environ Microbiol 64:3264–3269PubMedGoogle Scholar
  21. Maidak BL, Olsen GL, Larsen N, Overbeek R, McCaughey MJ, Woese CR (1997) The RDP (Ribosomal Database Project). Nucleic Acids Res 25:109–110PubMedGoogle Scholar
  22. Manz W, Eisenbrecher M, Neu TR, Szewzyk U (1998) Abundance and spatial organization of gram-negative sulfate-reducing bacteria in activated sludge investigated by in situ probing with specific 16S rRNA targeted oligonucleotides. FEMS Microbiol Ecol 25:43–61CrossRefGoogle Scholar
  23. Meijer WG, Nienhuis-Kuiper ME, Hansen TA (1999) Fermentative bacteria from estuarine mud: phylogenetic position of Acidaminobacter hydrogenoformans and description of a new type of gram-negative, propionigenic bacterium as Propionibacter pelophilus gen. nov., sp. nov. Int J Syst Bacteriol 49:1039–1044PubMedGoogle Scholar
  24. Meyer RL, Larsen LH, Revsbech NP (2002) Microscale biosensor for measurement of volatile fatty acids in anoxic environments. Appl Environ Microbiol 68:1204–1210CrossRefPubMedGoogle Scholar
  25. Millero FJ, Hershey JP (1989) Thermodynamics and kinetics of hydrogen sulfide in natural waters. In: Saltzman ES, Cooper WJ (eds) Biogenic sulfur in the environment. American Society for Microbiology, Washington, D.C., pp 282–313Google Scholar
  26. Moyer CL, Dobbs FC, Karl DM (1994) Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active hydrothermal vent system, Loihi Seamount, Hawaii. Appl Environ Microbiol 60:871–879PubMedGoogle Scholar
  27. Muyzer G, Teske A, Wirsen CO, Jannasch HW (1995) Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164:165–172CrossRefPubMedGoogle Scholar
  28. Nielsen PH, Raunkjaer K, Norsker NH, Jensen MA, Hvitved-Jacobsen T (1992) Transformation of wastewater in sewer systems—a review. Water Sci Technol 25:17–31Google Scholar
  29. Okabe S, Hirata K, Ozawa Y, Watanabe Y (1996) Spatial microbial distributions of nitrifiers and heterotrophs in mixed population biofilms. Biotechnol Bioeng 50:24–35CrossRefGoogle Scholar
  30. Okabe S, Itoh T, Satoh H, Watanabe Y (1999) Analyses of spatial distribution of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms. Appl Environ Microbiol 65:5107–5116PubMedGoogle Scholar
  31. Postgate JR (1984) The sulphate-reducing bacteria, 2nd edn. Cambridge University Press, Cambridge, UKGoogle Scholar
  32. Rabus R, Fukui M, Wilkes H, Widdel F (1996) Degradative capacities and 16S rRNA-targeted whole cell hybridization of sulfate-reducing bacteria in an anaerobic environment culture utilizing alkylbenzenes from crude oil. Appl Environ Microbiol 62:3605–3613PubMedGoogle Scholar
  33. Ramsing NB, Kühl M, Jorgensen BB (1993) Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes. Appl Environ Microbiol 59:3840–3849PubMedGoogle Scholar
  34. Ramsing NB, Fossing H, Ferdelman TG, Andersen F, 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 62:1391–1404PubMedGoogle Scholar
  35. Revsbech NP (1989) An oxygen microelectrode with a guard cathode. Limnol Oceanogr 55:1907–1910Google Scholar
  36. Revsbech NP, Jorgensen BB (1986) Microelectrodes: their use in microbial ecology. Adv Microb Ecol 9:293–352Google Scholar
  37. Saito N, Nei M (1987) The neighbor-joining method: a new method for constructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  38. Santegoeds CM, Ferdelman TG, Muyzer G, de Beer D (1998) Structural and functional dynamics of sulfate-reducing populations in bacterial biofilms. Appl Environ Microbiol 64:3731–3739PubMedGoogle Scholar
  39. Santegoeds CM, Damgaard LR, Hesselink G, Zopfi J, Lens P, Muyzer G, deBeer D (1999) Distribution of sulfate-reducing and methanogenic bacteria in anaerobic aggregates determined by microsensor and molecular analyses. Appl Environ Microbiol 65:4618–4629PubMedGoogle Scholar
  40. Snaidr J, Amann R, Huber I, Ludwig W, Schleifer K-H (1997) Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl Environ Microbiol 63:2884–2896PubMedGoogle Scholar
  41. Sørensen J, Christensen D, Jørgensen BB (1981) Volatile fatty acid and hydrogen as substrates for sulfate-reducing bacteria in anaerobic marine sediment. Appl Environ Microbiol 42:5–11Google Scholar
  42. Teske A, Wawer C, Muyzer G, Ramsing NB (1996) Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments. Appl Environ Microbiol 62:1405–1415PubMedGoogle Scholar
  43. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  44. Vester F, Ingvorsen K (1998) Improved most-probable-number method to detect sulfate-reducing bacteria with natural media and a radiotracer. Appl Environ Microbiol 64:1700–1707PubMedGoogle Scholar
  45. 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 59:1520–1525PubMedGoogle Scholar
  46. Wen A, Fegan M, Hayward C, Chakraborty S, Sly LI (1999) Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong (1926) and Tamaoka et al. (1987)) gen. nov., comb. nov. Int J Syst Bacteriol 49:567–576PubMedGoogle Scholar
  47. Widdel F (1988) Microbiology and ecology of sulfate-and sulfur-reducing bacteria. In: Zehnder AJB (ed) Biology of anaerobic microorganisms. Wiley, New York, pp. 469–585Google Scholar
  48. Widdel F, Pfenning N (1982) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids II. Incomplete oxidation of propionate by Desulfobulbus propionicus gen. nov., sp. nov. Arch Microbiol 131:360–365Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Department of Urban and Environmental Engineering, Graduate School of EngineeringHokkaido University Kita-kuJapan
  2. 2.Department of Civil Engineering, Faculty of EngineeringHachinohe Institute of TechnologyMyo, HachinoheJapan

Personalised recommendations