Antonie van Leeuwenhoek

, Volume 107, Issue 5, pp 1135–1144 | Cite as

Moderate oxygen depletion as a factor favouring the filamentous growth of Sphaerotilus natans

  • Marina Seder-ColominaEmail author
  • Anne Goubet
  • Sébastien Lacroix
  • Guillaume Morin
  • Georges Ona-Nguema
  • Giovanni Esposito
  • Eric D. Van Hullebusch
  • Jean-Jacques Pernelle
Original Paper


Sphaerotilus natans is a neutrophilic iron-related sheath-forming filamentous microorganism that presents dual morphotype: single cells and ensheathed cells forming filaments. As S. natans has been proposed as a sorbent for inorganic pollutants and it is occasionally involved in bulking episodes, elucidating factors affecting its filamentous growth is of crucial interest. The purpose of this work was to evaluate the effect of dissolved oxygen (DO) as a factor affecting S. natans filamentation from single cells. A method to quantify S. natans in its filamentous and single-cell morphotypes, based on a differential filtration procedure coupled with quantitative real-time PCR, was developed here. Scanning Electron Microscopy was used to validate the filtration step. Under actively aerated conditions (DO maintained at 7.6 ± 0.1 mg l−1), S. natans grew mainly as single cells throughout the experiment, while a depletion in DO concentration (to ~3 mg l−1) induced its filamentous growth. Indeed, when oxygen was reduced the proportion of single cells diminished from 83.3 ± 5.9  to 14.3 ± 3.4 % while the filaments increased from 16.7 ± 5.9  to 85.7 ± 3.4 %. Our results suggest that oxygen plays a key role in S. natans filamentation and contribute to better understanding of the filamentous proliferation of this bacterium. In addition, the proposed method will be helpful to evaluate other factors favouring filamentous growth.


Filamentous bacteria Dissolved oxygen Sphaerotilus natans qPCR 



The authors would like to thank the European Commission for providing financial support through the Erasmus Mundus Joint Doctorate Programme ETeCoS3 (Environmental Technologies for Contaminated Solids, Soils and Sediments) under the Grant agreement FPA n 2010-0009. In addition, the authors would like to thank Dr. Karim Benzerara for fruitful discussion and Imène Esteve for her help with Scanning Electron Microscope analyses. The purchase of the Scanning Electron Microscope (SEM) facility of the Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC) was supported by Région Ile-de-France grant SESAME 2006 N°I-07-593/R, INSU-CNRS, INP-CNRS, University Pierre et Marie Curie–Paris 6, and by the French National Research Agency (ANR) Grant no. ANR-07-BLAN-0124-01. The Confocal Laser Scanning Microscope (CLSM) Zeiss Axiovert 200 M LSM 510 META and the CFX 96™ Real-Time PCR Detection System (Bio Rad) used in this work are part of the MIMOSE experimental platform, and were funded by the Région Ile-de-France.

Supplementary material

10482_2015_405_MOESM1_ESM.docx (907 kb)
Supplementary material 1 (DOCX 907 kb)


  1. Amann R, Binder B, Olson R, Chisholm S, Devereux R, Stahl D (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedCentralPubMedGoogle Scholar
  2. Blackall LL, Seviour EM, Bradford D, Rossetti S, Tandoi V, Seviour RJ (2000) “Candidatus Nostocoida limicola”, a filamentous bacterium from activated sludge. Int J Syst Evol Microbiol 50:703–709CrossRefPubMedGoogle Scholar
  3. Bouché J-P, Pichoff S (1998) On the birth and fate of bacterial division sites. Mol Microbiol 29:19–26CrossRefPubMedGoogle Scholar
  4. Claessen D, Rozen DE, Kuipers OP, Søgaard-Andersen L, van Wezel GP (2014) Bacterial solutions to multicellularity: a tale of biofilms, filaments and fruiting bodies. Nature Rev Microbiol 12:115–124CrossRefGoogle Scholar
  5. Contreras EM, Giannuzzi L, Zaritzky NE (2000) Growth kinetics of the microorganism Sphaerotilus natans in a model system of a food industry wastewater. Water Res 34:4455–4463CrossRefGoogle Scholar
  6. Contreras EM, Giannuzzi L, Zaritzky NE (2004) Use of image analysis in the study of competition between filamentous and non-filamentous bacteria. Water Res 38:2621–2630CrossRefPubMedGoogle Scholar
  7. Den Besten HMW, Mols M, Moezelaar R, Zwietering MH, Abee T (2009) Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol 75:4111–4119CrossRefGoogle Scholar
  8. Dias FF, Dondero NC, Finstein MS (1968a) Attached growth of Sphaerotilus and mixed populations in a continuous-flow apparatus. Appl Microbiol 16:1191–1199PubMedCentralPubMedGoogle Scholar
  9. Dias FF, Okrend H, Dondero NC (1968b) Calcium nutrition of Sphaerotilus growing in a continuos-flow apparatus. Appl Environ Microbiol 16:1364–1369Google Scholar
  10. Eikelboom DH (1975) Filamentous organisms observed in activated sludge. Water Res 9:365–388CrossRefGoogle Scholar
  11. Eikelboom D, Van Buijsen H. (1981) Microscopic sludge investigation manual. TNO—Research Institute for Environmental Hygiene. TNO Report A 94aGoogle Scholar
  12. Gaudy E, Wolfe RS (1961) Factors affecting filamentous growth of Sphaerotilus natans. Appl Microbiol 9:580–584PubMedCentralPubMedGoogle Scholar
  13. Gaval G, Pernelle J-J (2003) Impact of the repetition of oxygen deficiencies on the filamentous bacteria proliferation in activated sludge. Water Res 37:1991–2000CrossRefPubMedGoogle Scholar
  14. Gino E, Starosvetsky J, Kurzbaum E, Armon R (2010) Combined chemical-biological treatment for prevention/rehabilitation of clogged wells by an iron-oxidizing bacterium. Environ Sci Technol 44:3123–3129CrossRefPubMedGoogle Scholar
  15. Gridneva E, Chernousova E, Dubinina G, Akimov V, Kuever J, Detkova E, Grabovich M (2011) Taxonomic investigation of representatives of the genus Sphaerotilus: descriptions of Sphaerotilus montanus sp. nov., Sphaerotilus hippei sp. nov., Sphaerotilus natans subsp. natans subsp. nov. and Sphaerotilus natans subsp. sulfidivorans subsp. nov., and an emended description of the genus Sphaerotilus. Int J Syst Evol Microbiol 61:916–925CrossRefPubMedGoogle Scholar
  16. Hou Y, Zhang H, Miranda L, Lin S (2010) Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene. PLoS ONE 5:e9545CrossRefPubMedCentralPubMedGoogle Scholar
  17. Jassby D, Xiao Y, Schuler AJ (2014) Biomass density and filament length synergistically affect activated sludge settling: systematic quantification and modeling. Water Res 48:457–465CrossRefPubMedGoogle Scholar
  18. Jenkins D (1992) Towards a comprehensive model of activated sludge bulking and foaming. Water Sci Technol 25:215–230Google Scholar
  19. Jenkins D, Richard M, Daigger G. (2004) Manual of the causes and control of activated sludge bulking, foaming, and other solids separation problems. 3rd Edition. Lewis PublishersGoogle Scholar
  20. Kondo K, Takeda M, Ejimaa W, Kawasaki Y, Umezua T, Yamada M, Koizumi J, Mashima T, Katahirab M (2011) Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase. Inter J Biol Macromol. 48:256–262CrossRefGoogle Scholar
  21. Lodi A, Solisio C, Converti A, Del Borghi M (1998) Cadmium, zinc, copper, silver and chromium (III) removal from wastewaters by Sphaerotilus natans. Bioprocess Eng 19:197–203Google Scholar
  22. Manz W, Amann R, Ludwig W, Wagner M, Schleifer K (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600CrossRefGoogle Scholar
  23. Martins AMP, Pagilla K, Heijnen JJ, van Loosdrecht MCM (2004) Filamentous bulking sludge—a critical review. Water Res 38:793–817CrossRefPubMedGoogle Scholar
  24. Pagnanelli F, Esposito A, Toro L, Vegliò F (2003) Metal speciation and pH effect on Pb, Cu, Zn and Cd biosorption onto Sphaerotilus natans: langmuir-type empirical model. Water Res 37:627–633CrossRefPubMedGoogle Scholar
  25. Palm J, Parker D (1980) Relationship between organic loading, dissolved oxygen concentration and sludge settleability in the completely-mixed activated sludge process. J Water Pollut Control Fed 52:2484–2506Google Scholar
  26. Park S, Kim D-H, Lee J-H, Hur H-G (2014) Sphaerotilus natans encrusted with nanoball-shaped Fe(III) oxide minerals formed by nitrate-reducing mixotrophic Fe(II) oxidation. FEMS Microbiol Ecol. doi: 10.1111/1574-6941.12372 PubMedCentralPubMedGoogle Scholar
  27. Patziger M, Kainz H, Hunze M, Józsa J (2012) Influence of secondary settling tank performance on suspended solids mass balance in activated sludge systems. Water Res 46:2415–2424CrossRefPubMedGoogle Scholar
  28. Pellegrin V, Juretschko S, Wagner M, Cottenceau G (1999) Morphological and biochemical properties of a Sphaerotilus sp. isolated from paper mill slimes. Appl Environ Microbiol 65:156–162PubMedCentralPubMedGoogle Scholar
  29. Richard M, Hao O, Jenkins D (1985) Growth kinetics of Sphaerotilus species and their significance in activated sludge bulking. J Water Pollut Control Fed 57:68–81Google Scholar
  30. Rossetti S, Hildisch D, Christensson C, Del Dot T, Blackall LL, Tandoi V (1997) Isolation and identification of an Eikelboom type 1863 strain as Acinetobacter johnsonii. Water Res 31:657–660CrossRefGoogle Scholar
  31. Seder-Colomina M, Morin G, Benzerara K, Ona-Nguema G, Pernelle J-J, Esposito G, van Hullebusch ED (2014) Sphaerotilus natans, a neutrophilic iron-related sheath-forming bacterium: perspectives for metal remediation strategies. Geomicrobiol J 31:64–75CrossRefGoogle Scholar
  32. Silverstein J, Mines R, Sherrard J, Weber A, Aitken M (1990) Activated sludge. Res J Water Pollut Control Fed. 62:398–406Google Scholar
  33. Smith CJ, Nedwell DB, Dong LF, Osborn AM (2006) Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environ Microbiol 8:804–815CrossRefPubMedGoogle Scholar
  34. Spring S (2006) The genera Leptothrix and Sphaerotilus. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes, vol 5. Springer, New York, pp 758–777CrossRefGoogle Scholar
  35. Strom PF, Jenkins D (1984) Identification and significance of filamentous microorganisms in activated sludge. J Water Pollut Control Fed 56:449–459Google Scholar
  36. Suzuki T, Kanagawa T, Kamagata Y (2002) Identification of a gene essential for sheathed structure formation in Sphaerotilus natans, a filamentous sheathed bacterium. Appl Environ Microbiol 68:365–371CrossRefPubMedCentralPubMedGoogle Scholar
  37. Takeda M, Miyanoiri Y, Nogami T, Oda K, Saito T, Kato K, Koizumi J, Katahira M (2007) Structural analysis of the fundamental polymer of the sheath constructed by Sphaerotilus natans. Biosci Biotechnol Biochem 71:2992–2998CrossRefPubMedGoogle Scholar
  38. Tanaka H, Kurano N, Ueda S, Ueda S, Okazaki M, Miura Y (1985) Model system of bulking and flocculation in mixed culture of Sphaerotilus sp. and Pseudomonas sp. for dissolved oxygen deficiency and high loading. Water Res 19:563–571CrossRefGoogle Scholar
  39. Tomei MC, Levantesi C, Rossetti S, Tandoi V (1999) Microbiological characterisation of pure cultures and its relevance to modelling and control of bulking phenomena. Water Sci Technol 39:21–29CrossRefGoogle Scholar
  40. Van Veen WL, Mulder EG, Deinema MH (1978) The Sphaerotilus-Leptothrix group of bacteria. Microbiol Rev 42:329–356PubMedCentralPubMedGoogle Scholar
  41. Wagner M, Amann R, Kämpfer P, Assmus B, Hartmann A, Hutzler P, Springer N, Schleifer KH (1994) Identification and in situ detection of gram-negative filamentous bacteria in activated sludge. Syst Appl Microbiol 17:405–417CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Marina Seder-Colomina
    • 1
    • 2
    Email author
  • Anne Goubet
    • 3
  • Sébastien Lacroix
    • 3
    • 5
  • Guillaume Morin
    • 4
  • Georges Ona-Nguema
    • 4
  • Giovanni Esposito
    • 2
  • Eric D. Van Hullebusch
    • 1
  • Jean-Jacques Pernelle
    • 3
  1. 1.Laboratoire Géomatériaux et Environnement (LGE) (EA 4508), Institut Francilien des Sciences AppliquéesUniversité Paris-Est UPEMMarne-la-vallée, Cedex 2France
  2. 2.Department of Civil and Mechanical EngineeringUniversity of Cassino and Southern LazioCassinoItaly
  3. 3.Institut national de recherche en sciences et technologies pour l’environnement et l’agriculture (Irstea)UR HBANAntony CedexFrance
  4. 4.Institut de Minéralogie, de Physique des Matériaux, et de Cosmochimie (IMPMC)UMR 7590, CNRS, UPMC, MNHN, IRDParis, Cedex 05France
  5. 5.Veolia Environnement Recherche and Innovation - Pôle Biotechnologies et AgronomieCentre de Recherche Veolia Environnement de Maisons LaffitteMaisons - LaffitteFrance

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