Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans

  • Sofia Andersson
  • Gunnel Dalhammar
  • Carl Johan Land
  • Gunaratna Kuttuva RajaraoEmail author
Applied Microbial and Cell Physiology


Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3–37%), nucleic acids (9–50%), and carbohydrates (3–21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.


Biofilm Comamonas denitrificans GC–MS Extracellular polymeric substances HPAEC Polysaccharides 



The authors are grateful to Gustav Sundqvist, Jens Eklöf, and Associate Professor Qi Zhou at the Department of Wood Biotechnology, School of Biotechnology, KTH, for their support with chromatographic instruments and Kaj Kauko for assistance with SEM. Special thanks to Associate Professor Harry Brumer and Professor Vincent Bulone for valuable discussions.


  1. Andersson S, Kuttuva Rajarao G, Land CJ, Dalhammar G (2008) Biofilm formation and interactions of bacterial strains found in wastewater treatment systems. FEMS Microbiol Lett 283:83–90CrossRefGoogle Scholar
  2. Beatson SA, Minamino T, Pallen MJ (2006) Variation in bacterial flagellins: from sequence to structure. Trends Microbiol 14:151–155CrossRefGoogle Scholar
  3. Beech I, Hanjagsit L, Kalaji M, Neal AL, Zinkevich V (1999) Chemical and structural characterization of exopolymers produced by Pseudomonas sp. NCIMB 2021 in continuous culture. Microbiology 145:1491–1497CrossRefGoogle Scholar
  4. Biermann CJ (1988) Analysis of carbohydrates by GLC and MS. CRC-Taylor & Francis, FloridaGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Böckelmann U, Janke A, Kuhn R, Neu TR, Wecke J, Lawrence JR, Szewzyk U (2006) Bacterial extracellular DNA forming a defined network-like structure. FEMS Microbiol Lett 262:31–38CrossRefGoogle Scholar
  7. Böckelmann U, Lünsdorf H, Szewzyk U (2007) Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate. Environ Microbiol 9:2137–2144CrossRefGoogle Scholar
  8. Comte S, Guibaud G, Baudu M (2007) Effect of extraction method on EPS from activated sludge: An HPSEC investigation. J Hazard Mater 140:129–137CrossRefGoogle Scholar
  9. Dove A (2006) News feature: drugs down the drain. Nat Med 12:376–377CrossRefGoogle Scholar
  10. Flemming H-C, Wingender J (2002) Extracellular polymeric substances (EPS): structural, ecological and technical aspects. In: Bitton G (ed) Encyclopedia of environmental microbiology. Wiley, New York, pp 1223–1231Google Scholar
  11. Flemming H-C, Neu TR, Wozniak DJ (2007) The EPS matrix: the “house of biofilm cells”. J Bacteriol 189:7945–7947CrossRefGoogle Scholar
  12. Frings CS, Fendley TW, Dunn RT, Queen CA (1972) Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. Clin Chem 18:673–674Google Scholar
  13. Gao B, Zhu X, Xu C, Yue Q, Li W, Wei J (2008) Influence of extracellular polymeric substances on microbial activity and cell hydrophobicity in biofilms. J Chem Technol Biotechnol 83:227–232CrossRefGoogle Scholar
  14. Guerry P (2007) Campylobacter flagella: not just for motility. Trends Microbiol 15:456–461CrossRefGoogle Scholar
  15. Gumaelius L, Magnusson G, Pettersson B, Dalhammar G (2001) Comamonas denitrificans sp. nov., an efficient denitrifying bacterium isolated from activated sludge. Int J Syst Evol Microbiol 51:999–1006Google Scholar
  16. Gumaelius L, Smith EH, Dalhammar G (1996) Potential biomarker for denitrification of wastewaters: effects of process variables and cadmium toxicity. Wat Res 30:3025–3031CrossRefGoogle Scholar
  17. Kachlany SC, Levery SB, Kim JS, Reuhs BL, Lion LW, Ghiorse WC (2001) Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7. Environ Microbiol 3:774–784CrossRefGoogle Scholar
  18. Kives J, Orgaz B, Sanjose C (2006) Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52. Colloids Surf B Biointerfaces 52:123–127CrossRefGoogle Scholar
  19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  20. Liu H, Fang HHP (2002) Extraction of extracellular polymeric substances (EPS) of sludges. J Biotechnol 95:249–256CrossRefGoogle Scholar
  21. Liu H-H, Yang Y-R, Shen X-C, Zhang Z-L, Shen P, Xie Z-X (2008) Role of DNA in bacterial aggregation. Curr Microbiol 57:139–144CrossRefGoogle Scholar
  22. Mandal SM, Ray B, Dey S, Pati BR (2007) Production and composition of extracellular polysaccharide synthesized by a Rhizobium isolate of Vigna mungo (L.) Hepper. Biotechnol Lett 29:1271–1275CrossRefGoogle Scholar
  23. Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S.-I, Lee YC (2005) Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 339:69–72CrossRefGoogle Scholar
  24. McConville MJ, Homans SW, Thomas-Oates JE, Dell A, Bacic A (1990) Structures of the glycoinositolphospholipids from Leishmania major. A family of novel galactofuranose-containing glycolipids. J Biol Chem 265:7385–7394Google Scholar
  25. Qureshi N, Annous B, Ezeji T, Karcher P, Maddox I (2005) Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates. Microbial Cell Factories 4:24CrossRefGoogle Scholar
  26. Rodgers M, Zhan XM (2003) Moving-medium biofilm reactors. Rev Environ Sci Biotechnol 2:213–224CrossRefGoogle Scholar
  27. Romaní A, Fund K, Artigas J, Schwartz T, Sabater S, Obst U (2008) Relevance of polymeric matrix enzymes during biofilm formation. Microb Ecol 56(3):427–436. doi: 10.1007/s00248-00007-09361-00248 CrossRefGoogle Scholar
  28. Sawardeker JS, Sloneker JH, Jeanes A (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal Chem 37:1602–1604CrossRefGoogle Scholar
  29. Schwarzenbach RP, Escher BI, Fenner K, Hofstetter TB, Johnson CA, von Gunten U, Wehrli B (2006) The challenge of micropollutants in aquatic systems. Science 313:1072–1077CrossRefGoogle Scholar
  30. Sheng G-P, Zhang M-L, Yu H-Q (2008) Characterization of adsorption properties of extracellular polymeric substances (EPS) extracted from sludge. Colloids Surf B 62:83–90CrossRefGoogle Scholar
  31. Späth R, Flemming HC, Wuertz S (1998) Sorption properties of biofilms. Water Sci Technol 37:207–210CrossRefGoogle Scholar
  32. Stewart PS (2006) Matrix mysteries hold keys to controlling biofilms. In: Biofilms Perspectives, February. Available at Accessed 16 Dec 2008
  33. Stewart PS, Franklin MJ (2008) Physiological heterogeneity in biofilms. Nat Rev Microbiol 6:199–210CrossRefGoogle Scholar
  34. Sutherland IW (1990) Biotechnology of microbial exopolysaccharides. Cambridge University Press, New YorkGoogle Scholar
  35. Talaga P, Vialle S, Moreau M (2002) Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. Vaccine 20:2474–2484CrossRefGoogle Scholar
  36. Ternes T (2007) The occurrence of micopollutants in the aquatic environment: a new challenge for water management. Water Sci Technol 55:327–332Google Scholar
  37. Toutain CM, Caizza NC, Zegans ME, O’Toole GA (2007) Roles for flagellar stators in biofilm formation by Pseudomonas aeruginosa. Res Microbiol 158:471–477CrossRefGoogle Scholar
  38. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295:1487CrossRefGoogle Scholar
  39. Wilderer PA, McSwain BS (2004) The SBR and its biofilm application potentials. Water Sci Technol 50:1–10Google Scholar
  40. Wuertz S, Spaeth R, Hinderberger A, Griebe T, Flemming HC, Wilderer PA (2001) A new method for extraction of extracellular polymeric substances from biofilms and activated sludge suitable for direct quantification of sorbed metals. Water Sci Technol 43:25–31Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Sofia Andersson
    • 1
  • Gunnel Dalhammar
    • 1
  • Carl Johan Land
    • 1
  • Gunaratna Kuttuva Rajarao
    • 1
    • 2
    Email author
  1. 1.Division of Environmental Microbiology, School of BiotechnologyRoyal Institute of Technology, AlbaNova University CentreStockholmSweden
  2. 2.KTH-BIO, AlbaNova University CentreStockholmSweden

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