Antonie van Leeuwenhoek

, Volume 96, Issue 2, pp 247–258 | Cite as

From industrial sites to environmental applications with Cupriavidus metallidurans

  • Ludo DielsEmail author
  • Sandra Van Roy
  • Safyih Taghavi
  • Rob Van Houdt
Original Paper


Cupriavidus metallidurans CH34 and related strains are adapted to metal contaminated environments. A strong resistance to environmental stressors and adaptation make it ideal strains for survival in decreasing biodiversity conditions and for bioaugmentation purposes in environmental applications. The soil bacterium C. metallidurans is able to grow chemolithoautotrophically on hydrogen and carbon dioxide allowing a strong resilience under conditions lacking organic matter. The biofilm growth on soil particles allows coping with starvation or bad conditions of pH, temperature and pollutants. Its genomic capacity of two megaplasmids encoding several heavy metal resistance operons allowed growth in heavy metal contaminated habitats. In addition its specific siderophores seem to play a role in heavy metal sequestration besides their role in the management of bioavailable iron. Efflux ATPases and RND systems pump the metal cations to the membrane surface where polysaccharides serve as heavy metal binding and nucleation sites for crystallisation of metal carbonates. These polysaccharides contribute also to flotation under specific conditions in a soil-heavy metals–bacteria suspension mixture. An inoculated moving bed sand filter was constructed to treat heavy metal contaminated water and to remove the metals in the form of biomass mixed with metal carbonates. A membrane based contactor allowed to use the bacteria as well in a versatile wastewater treatment system and to grow homogeneously formed heavy metal carbonates. Its behaviour toward heavy metal binding and flotation was combined in a biometal sludge reactor to extract and separate heavy metals from metal contaminated soils. Finally its metal-induced heavy metal resistance allowed constructing whole cell heavy metal biosensors which, after contact with contaminated soil, waste, solids, minerals and ashes, were induced in function of the bioavailable concentration (Cd, Zn, Cu, Cr, Co, Ni, Tl, Pb and Hg) in the solids and allowed to investigate the speciation of immobilization of those metals.


Anaerobic reactors Bioavailability Biosensors Cupriavidus metallidurans Heavy metals Heavy metal resistance Moving bed sand filter Soil remediation Water treatment 



Cellular dry matter


Metal resistance, nodulation and cell division


Metal removal by sand filter inoculation


Bacteria immobilized composite membrane reactor


3-Chlorobenzoate, trichloroethylene


Polyaromatic hydrocarbons

Cop D

Copper resistance protein D

Supplementary material

10482_2009_9361_MOESM1_ESM.pdf (16 kb)
Supplementary material 1 (PDF 17 kb)


  1. Bulich AA, Isenberg DL (1981) Use of the luminescent bacterial system for the rapid assessment of aquatic toxicity. ISA Trans 20:29–33PubMedGoogle Scholar
  2. Coenye T, Spilker T, Reik R, Vandamme P, Lipuma JJ (2005) Use of PCR analyses to define the distribution of Ralstonia species recovered from patients with cystic fibrosis. J Clin Microbiol 43:3463–3466PubMedCrossRefGoogle Scholar
  3. Collard JM, Corbisier P, Diels L, Dong Q, Jeanthon C, Mergeay M, Taghavi S, van der Lelie D, Wilmotte A, Wuertz S (1994) Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications. FEMS Microbiol Rev 14:405–414PubMedCrossRefGoogle Scholar
  4. Corbisier P, Thiry E, Diels L (1996) Bacterial biosensors for the toxicity assessment of solid wastes. Environ Tox Water Qual 11:171–177CrossRefGoogle Scholar
  5. Corbisier P, van der Lelie D, Borremans B, Provoost A, de Lorenzo V, Brown NL, Lloyd JR, Hobman JL, Csöregi E, Johansson G, Mattiason B (1999) Whole cell-protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal Clin Acta 387:235–244CrossRefGoogle Scholar
  6. Corbisier P, Diels L, Illangasekare T, Reible D, Reinhard M, Vangronsveld J (2002) Mobility and availability of contaminants. In: Reible D, Demnerova K (eds) Innovative appraoches to the on-site assessment, remediation of contaminated sites. Kluwer, The Netherlands, pp 31–65Google Scholar
  7. Diels L (1997) Heavy metal bioremediation of soil. In: Sheehan D (ed) Methods in biotechnology, vol 2: bioremediation protocols. Humana press, Totowa, pp 283–295Google Scholar
  8. Diels L, Mergeay M (1990) DNA probe mediated detection of resistant bacteria from soils highly polluted by heavy metals. Appl Environ Microbiol 56:1485–1491PubMedGoogle Scholar
  9. Diels L, Sadouk A, Mergeay M (1989) Large plasmids governing multiple resistances to heavy metals: a genetic approach. Toxicol Environ Chem 23:79–89CrossRefGoogle Scholar
  10. Diels L, Carpels M, Geuzens P, Mergeay M, Rymen T (1992) Method and device for cleaning soil polluted by at least one heavy metal. European Patent 92,203,049.9Google Scholar
  11. Diels L, Van Roy S, Mergeay M, Doyen W, Taghavi S, Leysen R (1993) Immobilization of bacteria in composite membranes and development of tubular membrane reactors for heavy metal recuperation. In Third international conference on effective membrane processes—New perspectives, Paterson R (ed), BHR Group Conference Series, Publication no. 3, 275–293Google Scholar
  12. Diels L, Dong Q, van der Lelie D, Baeyens W, Mergeay M (1995a) The czc operon of Alcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals. J Industr Microbiol 124:142–153CrossRefGoogle Scholar
  13. Diels L, Van Roy S, Somers K, Willems I, Doyen W, Mergeay M, Springael D, Leysen R (1995b) The use of bacteria immobilized in tubular membrane reactors for heavy metal recovery and degradation of chlorinated aromatics. J Memb Sci 100:249–258CrossRefGoogle Scholar
  14. Diels L, Van Roy S, Dong Q, Dresselaers T, Hennen A, Ryngaert A, Peys K, Springael D (1996a) Molecular approaches in biofilm studies of membrane reactors. Med Fac Landbouww Univ Gent 61/4b:1917–1924Google Scholar
  15. Diels L, Van Roy S, Leysen R, Mergeay M (1996b) Heavy metal bioprecipitation by Alcaligenes Eutrophus CH34 immobilized in a membrane bioreactor. Intern Biodet Biodegr 37:239CrossRefGoogle Scholar
  16. Diels L, De Smet M, Hooyberghs L, Corbisier P (1999) Heavy metals bioremediation of soil. Molec Biotech 12:149–158CrossRefGoogle Scholar
  17. Diels L, Spaans PH, Van Roy S, Hooyberghs L, Wouters H, Walter E, Winters J, Macaskie L, Finlay J, Pernfuss B, Woebking H, Pümpel T (2003) Heavy metals removal by sand filters inoculated with metal sorbing and precipitating bacteria. Hydrometallurgy 71:235–241CrossRefGoogle Scholar
  18. Dong Q, Mergeay M (1994) Czc/cnr efflux: a three-component chemiosmotic antiport pathway with a 12-transmembrane-helix protein. Mol Microbiol 14:185–187PubMedCrossRefGoogle Scholar
  19. Gilis A, Khan AM, Cornelis P, Meyer JM, Mergeay M, van der Lelie D (1996) Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric-alcaligin E receptor. J Bacteriol 178:5499–5507PubMedGoogle Scholar
  20. Höfte M, Dong Q, Kourambos S, Krishnapillai V, Sherratt D, Mergeay M (1994) The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site-specific recombinase. Mol Microbiol 14:1011–1020PubMedCrossRefGoogle Scholar
  21. Kefala MI, Zouboulis AI, Matis KA (1999) Biosorption of cadmium ions by Actinomycetes and separation by flotation. Environ Pollut 104:283–293CrossRefGoogle Scholar
  22. Leysen R, Doyen W(1987) European patent specification for Zirfon membranes. EP0241995 (B1)Google Scholar
  23. Liu YG, Huang N (1998) Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR. Plant Mol Biol Rep 16:175–181CrossRefGoogle Scholar
  24. Magrisso S, Erel Y, Belkin S (2008) Microbial reporters of metal bioavailability. Microb Biotechnol 1:320–330CrossRefGoogle Scholar
  25. Mergeay M (1991) Towards an understanding of the genetics of bacterial metal resistance. Trends Biotechnol 9:17–24PubMedCrossRefGoogle Scholar
  26. Mergeay M (2000) Bacteria adapted to industrial biotopes: the metal resistant Ralstonia. In: GSaR Hengge-Aronis (ed) Bacterial stress responses. ASM Press, Washington D.C., pp 403–414Google Scholar
  27. Mergeay M, Houba C, Gerits J (1978) Extrachromosomal inheritance controlling resistances to Cd++, Zn++, and Co++ ions: evidence from curing in a Pseudomonas. Arch Intern Physiol Bioch 86:440–441Google Scholar
  28. Mergeay M, Nies D, Schlegel G, Gerits J, Van Gijsegem F (1985) Alcaligenes eutrophus CH34, a facultative chemolithotroph displaying plasmid bound resistance to heavy metals. J Bacteriol 162:328–334PubMedGoogle Scholar
  29. Mergeay M, Sadouk A, Diels L, Faelen M, Gerits J, Denecke J, Powell B (1987) High level spontaneous mutagenesis revealed by survival at non-optimal temperature in Alcaligenes eutrophus CH34. Arch Inter Physiol Bioch 95:35–36Google Scholar
  30. Mergeay M, Monchy S, Vallaeys T, Auquier V, Benotmane A, Bertin P, Taghavi S, Dunn J, van der Lelie D, Wattiez R (2003) Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. FEMS Microb Rev 27:385–410CrossRefGoogle Scholar
  31. Mergeay M, Monchy S, Janssen P, Van Houdt R, Leys N (2009) Megaplasmids in Cupriavidus genus and metal resistance. In: Schwartz E (ed) Microbial megaplasmids. Springer, Berlin, p 320 ISBN: 978-3-540-85466-1Google Scholar
  32. Monchy S, Benotmane MA, Wattiez R, van Aelst S, Auquier V, Borremans B, Mergeay M, Taghavi S, van der Lelie D, Vallaeys T (2006) Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34. Microbiology 152:1765–1776PubMedCrossRefGoogle Scholar
  33. Monchy S, Benotmane MA, Janssen P, Vallaeys T, Taghavi S, van der Lelie D, Mergeay M (2007) Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans are specialized in the maximal viable response to heavy metals. J Bacteriol 189:7417–7425PubMedCrossRefGoogle Scholar
  34. Peys K, Diels L, Leysen R, Vandecasteele C (1997) Development of a membrane biofilm reactor for the degradation of chlorinated aromatics. Water Sci Tech 36:205–214CrossRefGoogle Scholar
  35. Podda F, Zuddas P, Minacci A, Pepi M, Baldi F (2000) Heavy metal co-precipitation with hydrozincite [Zn(5)(CO(3))(2)(OH)(6)] from mine waters caused by photosynthetic microorganisms. Appl Environ Microbiol 11:5092–5098CrossRefGoogle Scholar
  36. Pümpel T, Ebner C, Pernfuss B, Schinner F, Diels L, Keszthelyi Z, Macaskie L, Tsezos M, Wouters H (2001) Removal of nickel from plating rinsing water by a moving-bed sandfilter inoculated with metal sorbing and precipitating bacteria. Hydrometallurgy 59:383–393CrossRefGoogle Scholar
  37. Ruttens A, Mench M, Colpaert JV, Boisson J, Carleer R, Vangronsveld K (2006) Phytostabilization of a metal contaminated sandy soil. I: Influence of compost and/or inorganic metal immobilizing soil amendments on phytotoxicity and plant availability of metals. Environ Poll 144:524–532CrossRefGoogle Scholar
  38. Saier MH, Tam R, Reizer A, Reizer J (1994) Two novel families of bacterial membrane proteins concerned nodulation, cell division and transport. Mol Microbiol 11:841–847PubMedCrossRefGoogle Scholar
  39. Sato Y, Nishihara H, Yoshida M, Watanabe M, Rondal JD, Concepcion RN, Ohta H (2006) Cupriavidus pinatubonensis sp. nov. and Cupriavidus laharis sp. nov., novel hydrogen-oxidizing, facultatively chemolithotrophic bacteria isolated from volcanic mudflow deposits from Mt. Pinatubo in the Philippines. Int J Syst Evol Microbiol 56:973–978PubMedCrossRefGoogle Scholar
  40. Schultze-Lam S, Harauz G, Beveridge TS (1992) Participation of cyanobacterial S layer in fine grain mineral formation. J Bacteriol 174:7971–7981PubMedGoogle Scholar
  41. Shaw JJ, Settles LG, Kado CI (1988) Transposon Tn4431 mutagenesis of Xanthomonas campestris pv Campestris. Characterisation of a non-pathogenic mutant and cloning of a locus for pathogenicity. Mol Plant–Microbe Interact 1:39–45Google Scholar
  42. Springael D, Peys K, Ryngaert A, Van Roy S, Hooyberghs L, Ravatn R, Heyndrickx M, van der Meer JR, Vandecasteele C, Mergeay M, Diels L (2002) Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria. Environ Microbiol 4:70–80PubMedCrossRefGoogle Scholar
  43. Tesseir A, Campbell PGC, Bisson M (1979) Sequential extraction procedure for the speciation of particulate trace metals. Anal Chem 51:844–850CrossRefGoogle Scholar
  44. Tibazarwa C, Wuerz S, Mergeay M, Wyns L, van der Lelie D (2001) A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants. Environ Pollut 113:19–26PubMedCrossRefGoogle Scholar
  45. Van Assche F, Clijsters H (1990) A biological test system for the evaluation of the phytotoxicity of metal contaminated soils. Environ Pollut 66:157–172PubMedCrossRefGoogle Scholar
  46. Van der Lelie D, Verschaeve L, Regniers L, Corbisier P (2000) Use of bacterial tests (the VITOTOX (R) genotoxicity test and the BIOMET heavy metal test) to analyze chemicals and environmental samples. In: Personne G, Janssen C (eds) New microbiotests for routine toxicity screening and biomonitoring. Kluwer Academic Publishers, New York, pp 197–207Google Scholar
  47. Volesky B (2003) Biosorption process simulation tools. Hydrometallurgy 71:179−190CrossRefGoogle Scholar
  48. Woebking H, Diels L (2000) Abreicherung und Rückgewinnung von Eisen and Nichteisenmetallen aus industriellen Abwässeren unter Verwendung eines Bakterien geimpften Sandfilters. Berg- and Hüttenmännische Monatshefte 7:265–270Google Scholar
  49. Wuertz S, Mergeay M (1997) The impact of heavy metals on soil microbial communities and their activities. In: van Elsas D, Wellington E, Trevors J (eds) Modern soil microbiology. Marcel Dekker Publisher, New York, pp 607–642Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Ludo Diels
    • 1
    Email author
  • Sandra Van Roy
    • 1
  • Safyih Taghavi
    • 2
  • Rob Van Houdt
    • 3
  1. 1.BU Separation and Conversion TechnologyFlemish Institute for Technological Research (VITO)MolBelgium
  2. 2.Biology DepartmentBrookhaven National LaboratoryUptonUSA
  3. 3.Unit for Microbiology, Belgian Nuclear Research CentreSCK·CENMolBelgium

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