Journal of Soils and Sediments

, Volume 13, Issue 1, pp 176–188 | Cite as

Potential of willow and its genetically engineered associated bacteria to remediate mixed Cd and toluene contamination

  • Nele Weyens
  • Kerim Schellingen
  • Bram Beckers
  • Jolien Janssen
  • Reinhart Ceulemans
  • Daniel van der Lelie
  • Safiyh Taghavi
  • Robert Carleer
  • Jaco Vangronsveld



The purpose of this study was to investigate if bacteria with beneficial properties that were isolated from willow growing on a metal-contaminated site can be further equipped with genes coding for a specific degradation pathway to finally obtain transconjugants that can be inoculated in willow to improve phytoremediation efficiency of mixed contaminations.

Materials and methods

Cultivable rhizosphere bacteria and root endophytes were isolated from willow (cv. Tora) growing on a metal-contaminated soil. All isolated strains were tested for their metal resistance and potential to promote plant growth. The two most promising strains were selected and were equipped with the pTOM plasmid coding for toluene degradation. Both transconjugants were inoculated separately and combined in willow cuttings exposed to mixed Cd–toluene contamination, and their effect on phytotoxicity, Cd uptake, and toluene evapotranspiration was evaluated.

Results and discussion

Many of the isolated strains tested positive for the production of siderophores, organic acids, and indole acetic acid (IAA) and showed increased Cd resistance. The Cd-resistant, siderophore-producing rhizosphere strain Burkholderia sp. HU001 and the Cd-resistant root endophyte Pseudomonas sp. HU002, able to produce siderophores, organic acids, and IAA, were selected as receptors for conjugation with the toluene-degrading Burkholderia vietnamiensis BU61 as a donor of the pTOM-TCE plasmid. Although inoculation with the individual transconjugant strains had no effect on plant growth and negatively affected Cd uptake, their combined inoculation resulted in an increased shoot biomass upon Cd–toluene exposure did not affect Cd uptake and strongly reduced evapotranspiration of toluene to the atmosphere.


In this study, inoculation of willow with a consortium of plant-associated bacteria equipped with the appropriate characteristics resulted in an improved phytoremediation of a mixed Cd–toluene contamination: the degradation of toluene was improved leading to a decreased toxicity and evapotranspiration, while Cd uptake and translocation were not affected.


Cadmium Co contamination Plant-associated bacteria Salix Toluene Willow 



This research was funded by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) J.D. and by the Fund for Scientific Research Flanders (FWO-Vlaanderen), Ph.D. grant for S.C. and S.T. This work has been financially supported by the UHasselt Methusalem project 08M03VGRJ and by the European Commission under the Seventh Framwork Programme for Research (FP7-KBBE-266124, GREENLAND). S.T. was supported by Laboratory Directed Research and Development funds (LDRD09-005) at the Brookhaven National Laboratory under contract with the U.S. Department of Energy.


  1. Barac T, Taghavi S, Borremans B, Provoost A, Oeyen L, Colpaert JV, Vangronsveld J, van der Lelie D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nat Biotechnol 22:583–588CrossRefGoogle Scholar
  2. Barac T, Weyens N, Oeyen L, Taghavi S, van der Lelie D, Dubin D, Split M, Vangronsveld J (2009) Field note: hydraulic containment of a BTEX plume using poplar trees. Int J Phytoremediat 11:416–424CrossRefGoogle Scholar
  3. Braud A, Jézéquel K, Bazot S, Lebeau T (2009) Enhanced phytoextraction of an agricultural Cr-, Hg-, and Pb-contaminated soil by bioaugmentation with siderophore-producing bacteria. Chemosphere 74:280–286CrossRefGoogle Scholar
  4. Burkhardt C, Insam H, Hutchinson TC, Reber HH (1993) Impact of heavy metals on the degradative capabilities of soil bacterial communities. Biol Fertil Soils 16:154–156CrossRefGoogle Scholar
  5. Cunningham J, Kuiack C (1992) Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii. Appl Environ Microbiol 58:1451–1458Google Scholar
  6. Francis I, Holsters M, Vereecke D (2010) The Gram-positive side of plant–microbe interactions. Environ Microbiol 12:1–12CrossRefGoogle Scholar
  7. Glick BR, Todorovic B, Czarny J, Cheng Z, Duan J, McConkey B (2007) Promotion of plant growth by bacterial ACC deaminase. Crit Rev Plant Sci 26:227–242CrossRefGoogle Scholar
  8. Hogervorst J, Plusquin M, Vangronsveld J, Nawrot T, Cuypers A, Van Hecke E, Roels HA, Carleer R, Staessen JA (2007) House dust as possible route of environmental exposure to cadmium and lead in the adult general population. Environ Res 103:30–37CrossRefGoogle Scholar
  9. Kidd P, Barceló J, Bernal MP, Navari-Izzo F, Poschenrieder C, Shilev S, Clemente R, Monterosso C (2009) Trace elements behaviour at the root–soil interface: implications in phytoremediation. Environ Exp Bot 67:243–259CrossRefGoogle Scholar
  10. Koopmans GF, Römkens PFAM, Song J, Temminghoff EJM, Japenga J (2007) Predicting the phytoextraction duration to remediate heavy metal contaminated soils. Water Air Soil Pollut 181:355–371CrossRefGoogle Scholar
  11. Lebeau T, Braud A, Jézéquel K (2008) Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review. Environ Pollut 153:497–522CrossRefGoogle Scholar
  12. Li WC, Ye ZH, Wong MH (2007) Effects of bacteria on enhanced metal uptake of the Cd/Zn-hyperaccumulating plant, Sedum alfredii. J Exp Bot 58:4173–4182CrossRefGoogle Scholar
  13. Lin Q, Wang ZW, Ma S, Chen YX (2006) Evaluation of dissipation mechanisms by Lolium perenne L, and Raphanus sativus for pentachlorophenol (PCP) in copper co-contaminated soil. Sci Total Environ 368:814–822CrossRefGoogle Scholar
  14. Lodewyckx C, Taghavi S, Mergeay M, Vangronsveld J, Clijsters H, van der Lelie D (2001) The effect of recombinant heavy metal resistant endophytic bacteria on heavy metal uptake by their host plant. Int J Phytoremediat 3:173–187CrossRefGoogle Scholar
  15. Ma Y, Rajkumar M, Freitas H (2009) Improvement of plant growth and nickel uptake by nickel resistant-plant-growth promoting bacteria. J Hazard Mater 166:1154–1161CrossRefGoogle Scholar
  16. Mastretta C, Taghavi S, van der Lelie D, Mengoni A, Galardi F, Gonnelli C, Barac T, Boulet J, Weyens N, Vangronsveld J (2009) Endophytic bacteria from seeds of Nicotiana tabacum can reduce cadmium phytotoxicity. Int J Phytoremediat 11:251–267CrossRefGoogle Scholar
  17. Maxted AP, Black CR, West HM, Crout NMJ, McGrath SP, Young SD (2007) Phytoextraction of cadmium and zinc by Salix from soil historically amended with sewage sludge. Plant Soil 290:157–172CrossRefGoogle Scholar
  18. Meers E, Vandecasteele B, Ruttens A, Vangronsveld J, Tack FMG (2007) Potential of five willow species (Salix spp.) for phytoextraction of heavy metals. Environ Exp Bot 60:57–68CrossRefGoogle Scholar
  19. Mergeay M, Nies D, Schlegel HG, Gerits J, Charles P, Van Gijsegem F (1985) Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol 162:328–334Google Scholar
  20. Nawrot T, Plusquin M, Hogervorst J, Roels HA, Celis H, Thijs L, Vangronsveld J, Van Hecke E, Staessen JA (2006) Environmental exposure to cadmium and risk of cancer: a prospective population-based study. Lancet Oncol 7:119–126CrossRefGoogle Scholar
  21. Nawrot TS, Van Hecke E, Thijs L, Richart T, Kuznetsova T, Jin Y, Vangronsveld J, Roels HA, Staessen JA (2008) Cadmium-related mortality and long-term secular trends in the cadmium body burden of an environmentally exposed population. Environ Health Perspect 116:1620–1628CrossRefGoogle Scholar
  22. Patten C, Glick B (2002) Role of pseudomonas putida indoleacetic acid in development of the host plant root system. Appl Environ Microbiol 68:3795–3801CrossRefGoogle Scholar
  23. Rajkumar M, Ae N, Freitas H (2009) Endophytic bacteria and theit potential to enhance heavy metal phytoextraction. Chemosphere 77:153–160CrossRefGoogle Scholar
  24. Ruttens A, Boulet J, Weyens N, Smeets K, Adriaensen K, Meers E, Van Slycken S, Tack F, Meiresonne L, Thewys T, Witters N, Carleer R, Dupae J, Vangronsveld J (2011) Short rotation coppice culture of willow and poplar as energy crops on metal contaminated agriculture soils. Int J Phytoremediat 13:194–207CrossRefGoogle Scholar
  25. Said WA, Lewis DA (1991) Quantitative assessment of the effects of metals on microbial degradation of organic chemicals. Appl Environ Microbiol 57:1498–1503Google Scholar
  26. Sandrin TR, Maier RM (2003) Impact of metals on the biodegradation of organic pollutants. Environ Health Perspect 111:1093–1101CrossRefGoogle Scholar
  27. Saravanan VS, Madhaiyan M, Thangaraju M (2007) Solubilization of zinc compounds by the diazotrophic, plant growth promoting bacterium Gluconacetobacter diazotrophicus. Chemosphere 66:1794–1798CrossRefGoogle Scholar
  28. Schwyn B, Neilands J (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56CrossRefGoogle Scholar
  29. Sessitsch A, Puschenreiter M (2008) Endophytes and rhizosphere bacteria of plants growing in heavy metal-containing soils. In: Dion P, Nautiyal CS (eds) Microbiology of extreme soils. Springer, Berlin, pp 317–332CrossRefGoogle Scholar
  30. Sheng X-F, Xia J-J, Jiang C-Y, He L-Y, Qian M (2008) Characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape. Environ Pollut 156:1164–1170CrossRefGoogle Scholar
  31. Shields MS, Reagin MJ (1992) Selection of a Pseudomonas cepacia strain constitutive for the degradation of trichloroethylene. Appl Environ Microbiol 58:3977–3983Google Scholar
  32. Shields MS, Reagin MJ, Gerger RR, Campbell R, Somerville C (1995) TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4. Appl Environ Microbiol 61(4):1352–1356Google Scholar
  33. Siciliano SD, Fortin N, Mihoc A, Wisse G, Labelle S, Beaumier D, Ouelette D, Roy R, Whyte LG, Banks MK, Schwab P, Lee K, Greer CW (2001) Selection of specific endophytic bacterial genotypes by plants in response to soil contamination. Appl Environ Microbiol 67:2469–2475CrossRefGoogle Scholar
  34. Taghavi S, Barac T, Greenberg B, Borremans B, Vangronsveld J, van der Lelie D (2005) Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene. Appl Environ Microbiol 71:8500–8505CrossRefGoogle Scholar
  35. Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, van der Lelie D (2009) Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol 75:748–757CrossRefGoogle Scholar
  36. Thewys T, Witters N, Van Slycken S, Ruttens A, Meers E, Tack FMG, Vangronsveld J (2010a) Economic viability of phytoremediation of a cadmium contaminated agricultural area using energy maize part I: impact on the farmer's income. Int J Phytoremediat 12(7):650–662CrossRefGoogle Scholar
  37. Thewys T, Witters N, Meers E (2010b) Economic viability of phytoremediation of a cadmium contaminated agricultural area using energy maize part II: economics of anaerobic digestion of heavy metal contaminated maize in Belgium. Int J Phytoremediat 12(7):663–679CrossRefGoogle Scholar
  38. Top E, Van Rollegem P, van der Lelie D, Mergeay M, Verstraete W (1992) The importance of retromobilization to gene dissemination. In: Gauthier MJ (ed) Gene Transfers and Environment. Springer-Verlag Heidelberg, FRG, pp 127–134Google Scholar
  39. Valls M, de Lorenzo V (2002) Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiol Rev 26:327–338Google Scholar
  40. van der Lelie D (1998) Biological interactions: the role of soil bacteria in the bioremediation of heavy metal-polluted soils. In: Vangronsveld J, Cunningham SD (eds) Metal-contaminated soils: in situ inactivation and phytorestoration. Springer, Berlin Heidelberg New York, pp 31–50Google Scholar
  41. van der Lelie D, Schwitzguébel J-P, Vangronsveld J, Baker AJM (2001) Assessing phytoremediation's progress in the United States and Europe. Environ Sci Technol 35:446A–452ACrossRefGoogle Scholar
  42. van der Lelie D, Taghavi S, Monchy S, Schwender J, Miller L, Ferrieri R, Rogers A, Wu X, Zhu W, Weyens N, Vangronsveld J, Newman L (2009) Poplar and its bacterial endophytes: coexistence and harmony. Crit Rev Plant Sci 28:346–358CrossRefGoogle Scholar
  43. Van Ginneken L, Meers E, Guisson R, Ruttens A, Elst K, Tack FMG, Vangronsveld J, Diels L, Dejonghe W (2007) Phytoremediation for heavy metal contaminated soils combined with bioenergy production. J Environ Eng Landsc 15:227–236Google Scholar
  44. Vangronsveld J, Herzig R, Weyens N, Boulet J, Adriaensen K, Ruttens A, Thewys T, Vassilev A, Meers E, Nehnevajova E, van der Lelie D, Mench M (2009) Phytoremediation of contaminated soils and groundwater: lessons from the field. Environ Sci Pollut Res 16:765–794CrossRefGoogle Scholar
  45. Weyens N, van der Lelie D, Taghavi S, Vangronsveld J (2009a) Phytoremediation: plant–endophyte partnerships take the challenge. Curr Opin Biotechnol 20:248–254CrossRefGoogle Scholar
  46. Weyens N, van der Lelie D, Taghavi S, Newman L, Vangronsveld J (2009b) Exploiting plant–microbe partnerships for improving biomass production and remediation. Trends Biotechnol 27:591–598CrossRefGoogle Scholar
  47. Weyens N, Taghavi S, Barac T, van der Lelie D, Boulet J, Artois T, Carleer R, Vangronsveld J (2009c) Bacteria associated with oak and Ash on a TCE-contaminated site: characterization of isolates with potential to avoid evapotranspiration. Environ Sci Pollut Res 16:830–843CrossRefGoogle Scholar
  48. Weyens N, van der Lelie D, Artois T, Smeets K, Taghavi S, Newman L, Carleer R, Vangronsveld J (2009d) Bioaugmentation with engineered endophytic bacteria improves contaminant fate in phytoremediation. Environ Sci Technol 43:9413–9418CrossRefGoogle Scholar
  49. Weyens N, Truyens S, Dupae J, Newman L, van der Lelie D, Carleer R, Vangronsveld J (2010a) Potential of Pseudomonas putida W619-TCE to reduce TCE phytotoxicity and evapotranspiration in poplar cuttings. Environ Pollut 158:2915–2919CrossRefGoogle Scholar
  50. Weyens N, Truyens S, Saenen E, Boulet J, Dupae J, van der Lelie D, Carleer R, Vangronsveld J (2010b) Endophytes and their potential to deal with co-contamination of organic contaminants (toluene) and toxic metals (nickel) during phytoremediation. Int J Phytoremediat 13:244–255CrossRefGoogle Scholar
  51. Weyens N, Croes S, Dupae J, van der Lelie D, Carleer R, Vangronsveld J (2010c) Endophytes to deal with co-contamination of Ni and TCE. Environ Pollut 158:2422–2427CrossRefGoogle Scholar
  52. Weyens N, Boulet J, Adriaensen D, Timmermans J-P, Prinsen E, Van Oevelen S, D’Haen J, Smeets K, van der Lelie D, Taghavi S, Vangronsveld J (2012) Contrasting colonization and plant growth promoting capacity between wild type and a gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar. Plant Soil 356(1–2):217–230CrossRefGoogle Scholar
  53. Yang J, Kloepper J, Ryu C-M (2009) Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci 14:1–4CrossRefGoogle Scholar
  54. Zhuang X, Chen J, Shim H, Bai Z (2007) New advances in plant growth-promoting rhizobacteria for bioremediation. Environ Int 33:406–413CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Nele Weyens
    • 1
  • Kerim Schellingen
    • 1
  • Bram Beckers
    • 1
  • Jolien Janssen
    • 1
  • Reinhart Ceulemans
    • 2
  • Daniel van der Lelie
    • 3
  • Safiyh Taghavi
    • 3
  • Robert Carleer
    • 1
  • Jaco Vangronsveld
    • 1
  1. 1.Centre for Environmental SciencesHasselt UniversityDiepenbeekBelgium
  2. 2.Biology Department, Campus Drie EikenUniversity of AntwerpWilrijkBelgium
  3. 3.Research Triangle Institute (RTI) InternationalResearch Triangle ParkUSA

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