Skip to main content
SpringerLink
Log in

Extraction of extracellular polymeric substances from extreme acidic microbial biofilms

  • Methods
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The efficiency of five extraction methods for extracellular polymeric substances (EPS) was compared on three benthic eukaryotic biofilms isolated from an extreme acidic river, Río Tinto (SW, Spain). Three chemical methods (MilliQ water, NaCl, and ethylenediamine tetraacetic acid [EDTA]) and two physical methods (Dowex 50.8 and Crown Ether cation exchange resins) were tested. The quality and quantity of the EPS extracted from acidic biofilms varied according to which EPS extraction protocol was used. Higher amounts were obtained when NaCl and Crown Ether resins were used as extractant agents, followed by EDTA, Dowex, and MilliQ. EPS amounts varied from approximately 155 to 478 mg g−1 of dry weight depending on the extraction method and biofilm analyzed. EPS were primarily composed of carbohydrate, heavy metals, and humic acid, plus small quantities of proteins and DNA. Neutral hexose concentrations corresponded to more than 90% of the total EPS dry weight. The proportions of each metals in the EPS extracted with EDTA are similar to the proportions present in the water from each locality where the biofilms were collected except for Al, Cu, Zn, and Pb. In this study, the extracellular matrix heavy metal sorption efficiencies of five methods for extracting EPS from eukaryotic acidic biofilms were compared.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Aguilera A, Manrubia SC, Gómez F, Rodríguez N, Amils R (2006) Eukaryotic community distribution and their relationship with the water physicochemical parameters in an extreme acidic environment, Río Tinto (SW, Spain). Appl Environ Microbiol 72:5325–5330

    CAS  PubMed  PubMed Central  Google Scholar 

  • Aguilera A, Souza-Egipsy V, Gómez F, Amils R (2007a) Development and structure of eukaryotic biofilms in an extreme acidic environment, Río Tinto (SW, Spain). Microb Ecol 53:294–305

    PubMed  Google Scholar 

  • Aguilera A, Zettler E, Gómez F, Amaral-Zettler L, Rodríguez N, Amils R (2007b) Distribution and seasonal variability in the benthic eukaryotic community of Río Tinto (SW, Spain), an acidic, high metal extreme environment. Syst Appl Microbiol 30:531–546

    CAS  PubMed  Google Scholar 

  • Allison DG, Sutherland IW (1987) Role of exopolysaccharides in adhesion of freshwater bacteria. J Gen Microbiol 133:1319–1327

    CAS  Google Scholar 

  • Amaral LA, Gómez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Eukaryotic diversity in Spain’s river of fire. Nature 417:137

    Google Scholar 

  • Beech IB, Sunner J (2004) Biocorrosion:towards understanding interactions between biofilms and metals. Curr Opin Biotechnol 15:181–186

    CAS  PubMed  Google Scholar 

  • Bitton G, Friehofer V (1978) Influence of extracellular polysaccharide on the toxicity of copper and cadmium toward Klebsiella aerogenes. Microb Ecol 4:119–125

    CAS  Google Scholar 

  • Brown MJ, Lester JN (1979) Metal removal in activated sludge: the role of bacterial extracellular polymers. Water Res 13:817–837

    CAS  Google Scholar 

  • Brown MJ, Lester JN (1980) Composition of bacterial extracellular polymer extraction methods. Appl Environ Microbiol 40:179–185

    CAS  PubMed  PubMed Central  Google Scholar 

  • Burton K (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–323

    CAS  PubMed  PubMed Central  Google Scholar 

  • Danese PN (2000) Exopolysaccharide production is required for development of Escherichia coli K12 biofilm architecture. J Bacteriol 182:3593–3596

    CAS  PubMed  PubMed Central  Google Scholar 

  • Decho AW (1990) Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes. Oceanogr Mar Biol Ann Rev 28:73–153

    Google Scholar 

  • De la Noue J, DePaw N (1988) The potential of microalgal biotechnology: a review of production and uses of microalgae. Biotechnol Adv 6:725–770

    PubMed  Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    CAS  Google Scholar 

  • Ernst WHO (1998) Effects of heavy metals in plants at cellular and organismic level. In: Schürmann G, Marker B (eds) Ecotoxicology. Wiley, New York, pp 587–620

    Google Scholar 

  • Ferris FG, Schultze S, Witten TC, Fyfe WS, Beveridge TJ (1989) Metal interactions with microbial biofilms in acidic and neutral pH environments. Appl Environ Microbiol 55:1249–1257

    CAS  PubMed  PubMed Central  Google Scholar 

  • Frolund B, Griebe T, Nielsen PH (1995) Enzymatic activity in the activated sludge floc matrix. Appl Microbiol Biotechnol 43:755–761

    CAS  PubMed  Google Scholar 

  • Frolund B, Palmgren R, Keiding K, Halkjaer P (1996) Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res 30:1749–1758

    Google Scholar 

  • García-Meza JV, Barrangue C, Admiraal W (2005) Biofilm formation by algae as a mechanism for surviving on mine tailings. Environ Toxicol Chem 24:573–581

    PubMed  Google Scholar 

  • Gehr R, Henry JG (1983) Removal of extracellular material, techniques and pitfalls. Water Res 17:1743–1748

    CAS  Google Scholar 

  • Gross W, Robbins EI (2000) Acidophilic and acid-tolerant fungi and yeast. Hydrobiologia 433:91–109

    Google Scholar 

  • Guibaud G, Tixier N, Bouju A, Baudu M (2003) Relation between extracellular polymers’ composition and its ability to complex Cd, Cu and Pb. Chemosphere 52:1701–1710

    CAS  PubMed  Google Scholar 

  • Hirst CN, Jordan IA (2003) Distribution of exopolymeric substances in the littoral sediments of an oligotrophic lake. Microb Ecol 46:22–32

    CAS  PubMed  Google Scholar 

  • Holding KL, Gill RA, Carter J (2003) The relationship between epilithic periphyton (biofilm) bound metals and metals bound to sediments in freshwater systems. Environ Geochem Health 25:87–93

    CAS  PubMed  Google Scholar 

  • Hynes HBN (1970) The ecology of running waters. Liverpool Univ. Press, Liverpool

    Google Scholar 

  • James RO, Healey TW (1972) Adsorption of hydrolysable metal ions at the oxide–water interface. III. A thermodynamic model of adsorption. J Colloid Interface Sci 40:65–81

    CAS  Google Scholar 

  • Jefree CE, Read ND (1991) Ambient and low-temperature scanning electron microscopy. In: Hall JL, Hawes C (eds) Electron microscopy of plant cells. Academic Press, London, pp 313–413

    Google Scholar 

  • Jenkinson HF, Lappin-Scott HM (2001) Biofilms adhere to stay. Trends Microbiol 9:9–10

    CAS  PubMed  Google Scholar 

  • Kaplan D, Christiaen D, Arad S (1987) Binding of heavy metals by algal polysaccharides. In: Stadler T, Mollion J, Verdus MC, Karamanos Y, Moran H Christiaen D (eds) Algal biotechnology. Elsevier, London, UK, pp 179–187

    Google Scholar 

  • Liu H, Fang HP (2002) Extraction of extracellular polymeric substances (EPS) of sludges. J Biotechnol 95:249–256

    CAS  PubMed  Google Scholar 

  • Lock MA (1993) Attached microbial communities in rivers. In: Ford TE (ed) Aquatic microbiology, an ecological approach. Blackwell, Oxford, pp 113–138

    Google Scholar 

  • López-Archilla AI, Marín I, Amils R (2001) Microbial community composition and ecology of an acidic aquatic environment: the Tinto river, Spain. Microbial Ecol 41:20–35

    Google Scholar 

  • Mancuso-Nichols CA, Guezennec J, Bowman JP (2005) Bacterial exopolysaccharides from extreme marine environments with special consideration of the southern ocean, sea ice, and deep-sea hydrothermal vents: a review. Mar Biotechnol 7:253–271

    Google Scholar 

  • Mazor G, Kidon GJ, Vonshak A, Abeliovich A (1996) The role of cyanobacterial exopolysaccharides in structuring desert microbial crusts. Microb Ecol 21:121–130

    CAS  Google Scholar 

  • McLean RJC, Beveridge TJ (1990) Metal-binding capacity of bacterial surfaces and their ability to form mineralized aggregates. In: Ehrlich HL, Brierley CL (eds) Microbial mineral recovery. McGraw-Hill, New York, pp 234–253

    Google Scholar 

  • Nixdorf B, Mischke U, Lessmann D (1998) Chrysophytes and Chlamydomonads: pioneer colonists in extremely acidic mining lakes (pH < 3) in Lusitania (Germany). Hydrobiologia 369/370:315–327

    CAS  Google Scholar 

  • Nordstrom DK, Alpers CN (1999) Negative pH, efflorescent mineralogy and consequences from environmental restoration at the Iron Mountain Superfund site, California. Proc Nat Acad Sci U S A 96:3455–3462

    CAS  Google Scholar 

  • Novak JT, Haugan BE (1981) Polymer extraction from activated sludge. J Water Pollut Control Fed 53:1420–1424

    CAS  Google Scholar 

  • Nowack B, Kari FG, Hilger SU, Sigg L (1996) Determination of dissolved and adsorbed EDTA species in water and sediments by HPLC. Anal Chem 68:561–566

    CAS  PubMed  Google Scholar 

  • Park C, Novak JT (2007) Characterization of activated sludge exocellular polymers using several cation-associated extraction methods. Water Res 41:1679 – 1688 DOI https://doi.org/10.1016/j.watres.2007.01.031

    CAS  PubMed  Google Scholar 

  • Platt RM, Geesey GG, Davis JD, White DC (1985) Isolation and chemical analysis of firmly bound exopolysaccharides from adherent cells of a fresh water sediment bacterium. Can J Microbiol 31:675–680

    CAS  PubMed  Google Scholar 

  • Rougeaux H, Guezennec M, Che LM, Payri C, Deslandes E, Guezennec J (2001) Microbial communities and exopolysaccharides from Polynesian mats. Mar Biotechnol 3(2):181–187

    CAS  Google Scholar 

  • Staats N, Winder BD, Stal LJ, Mur LR (1999) Isolation and characterization of extracellular polysaccharides from the epipelic diatoms Cylindrotheca closterium and Navilcula salinarum. Eur J Phycol 34:161–169

    Google Scholar 

  • Stone AT (1997) Reactions of extracellular organic ligands with dissolved metal ions and mineral surfaces. In: Banfield JF, Nealson KH (eds) Geomicrobiology: interactions between microbes and minerals. Reviews in Mineralogy, 35. Mineralogical Society of America, Washington, pp 309–334

    Google Scholar 

  • Sutherland IW (2001) The biofilm matrix, an immobilized but dynamic microbial environment. TRENDS Microbiol 9:222–227

    CAS  PubMed  Google Scholar 

  • Stumm W, Morgan JJ (1985) Aquatic chemistry. Wiley, New York

    Google Scholar 

  • Umrania VV (2006) Bioremediation of toxic heavy metals using acidophilic autotrophes. Biosource Technol 97:1237–1242

    CAS  Google Scholar 

  • Underwood GJC, Paterson DM, Parkes RJ (1995) The measurement of microbial carbohydrate exopolymers from intertidal sediments. Limnol Oceanogr 40(7):1243–1253

    CAS  Google Scholar 

  • Vierira R, Volesky B (2000) Biosorption: a solution to pollution. Internatl Microbiol 3:17–24

    Google Scholar 

  • Volesky B, Holan ZS (1995) Biosorption of heavy metals. Biotechnol Prog 45:11235–11250

    Google Scholar 

  • Winder B, Staats N, Stal LJ, Paterson DM (1999) Carbohydrate secretion by phototrophic communities in tidal sediments. J Sea Res 42:131–146

    Google Scholar 

  • 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 43:25–31

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The work has been funded by grants to the Centro de Astrobiología at the Instituto National de Técnica Aeroespacial and CGL2005-05470/BOS grant. MaP. Martín is acknowledged for the ICP-MS and TXRF analysis, and Fundación Río Tinto is gratefully recognized for logistical field support. Work by AA and VS-E was supported by the Spanish Ministry of Education and Science through the program Ramón y Cajal. P. San Martín is a CSIC fellow from the I3P program supported by the European Community.

Author information

Authors and Affiliations

  1. Centro de Astrobiología (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejón de Ardoz, 28850, Madrid, Spain

    Angeles Aguilera, Virginia Souza-Egipsy & Ricardo Amils

  2. Centro de Biología Molecular (UAM-CSIC), Universidad Autónoma, C/ Nicolás Cabrera 1, Campus de. Cantoblanco, 28049, Madrid, Spain

    Patxi San Martín-Úriz & Ricardo Amils

Authors
  1. Angeles Aguilera
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Virginia Souza-Egipsy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patxi San Martín-Úriz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ricardo Amils
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Angeles Aguilera.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aguilera, A., Souza-Egipsy, V., San Martín-Úriz, P. et al. Extraction of extracellular polymeric substances from extreme acidic microbial biofilms. Appl Microbiol Biotechnol 78, 1079–1088 (2008). https://doi.org/10.1007/s00253-008-1390-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1390-9

Keywords

Search

Navigation