Microbial Ecology

, Volume 49, Issue 3, pp 443–450 | Cite as

Culture Independent Detection of Sphingomonas sp. EPA 505 Related Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons (PAHs)

  • N.M. Leys
  • A. Ryngaert
  • L. Bastiaens
  • E.M. Top
  • W. Verstraete
  • D. SpringaelEmail author


The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 102 cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99–100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.


Polycyclic Aromatic Hydrocarbon Sphingomonas Sphingobium Strain HS122 Sphingomonas Strain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was supported by a Vito PhD-fellowship (N.M.L.) and the EC project QLRT-1999-00326. We thank S. Schioetz-Hansen, J. Vandenberghe and J. Amor for providing PAH contaminated soil samples and P. De Vos for providing Sphingomonas reference strains.


  1. 1.
    Adkins, A 1999Degradation of the phenoxy acid herbicide diclofop-methyl by Sphingomonas paucimobilis isolated from a Canadian prairie soilJ Ind Microbiol Biotechnol23332335Google Scholar
  2. 2.
    Altschul, SF, Gish, W, Miller, W, Myers, EW, Lipman, DJ 1990Basic local alignment search toolJ Mol Biol215403410Google Scholar
  3. 3.
    Balkwill, DL, Drake, GR, Reeves, RH, Frederickson, JK, White, DC, Ringelberg, DB, Chandler, DP, Romine, MF, Kennedy, DW, Spadoni, CM 1997Taxonomic study of aromatic-degrading bacteria from deep-terrestial-subsurface sediments and description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. novInt J Syst Bacteriol47191201Google Scholar
  4. 4.
    Barkay, T, Navon-Venezia, S, Ron, EZ, Rosenberg, E 1999Enhancement of solubilization and biodegradation of polyaromatic hydrocarbons by the bioemulsifier alasanAppl Environ Microbiol6526972702Google Scholar
  5. 5.
    Bastiaens, L (1998) Isolation and characterization of polycyclic aromatic hydrocarbon degrading bacteria and evaluation of the use of isolates in soil slurry experiments. PhD Thesis, Doctoraatsproefschrift nr 363, KULeuven, Faculteit Landbouwkundige en Toegepaste Biologische WetenschappenGoogle Scholar
  6. 6.
    Bastiaens, L, Springael, D, Wattiau, P, Harms, H, deWachter, R, Verachtert, H, Diels, L 2000Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriersAppl Environ Microbiol6618341843Google Scholar
  7. 7.
    Bastiaens, L, Springael, D, Dejonghe, W, Wattiau, P, Verachtert, H, Diels, L 2001A transcriptional luxAB reporter fusion responding to fluorene in Sphingomonas sp. LB126 and its characterisation for whole-cell bioreporter purposesRes Microbiol152849859Google Scholar
  8. 8.
    Cassidy, MB, Lee, H, Trevors, JT, Zablotowicz, RB 1999Chlorophenol and nitrophenol metabolism by Sphingomonas sp UG30J Ind Microbiol Biotechnol23232241Google Scholar
  9. 9.
    Christner, BC, Mosley-Thompson, E, Thompson, LG, Reeve, JN 2001Isolation of bacteria and 16S rDNAs from Lake Vostok accretion iceEnviron Microbiol3570577Google Scholar
  10. 10.
    Cole, JR, Chai, B, Marsh, TL, Farris, RJ, Wang, Q, Kulam, SA, Chandra, S, McGarrell, DM, Schmidt, TM, Garrity, GM, Tiedje, JM 2003The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomyNucleic Acids Res31442443Google Scholar
  11. 11.
    Crawford, RL, Ederer, MM 1999Phylogeny of a Sphingomonas sp. that degrades pentachlorophenolJ Ind Microbiol Biotechnol23320325Google Scholar
  12. 12.
    Fantroussi, S, Verschuere, L, Verstraete, W, Top, EM 1999Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profilesAppl Environ Microbiol65982988Google Scholar
  13. 13.
    Feng, X, Ou, L-T, Ogram, A 1997Plasmid-mediated mineralisation of carbofuran by Sphingomonas sp. strain CF06Appl Environ Microbiol6313321337Google Scholar
  14. 14.
    Fujii, K, Urano, N, Ushio, H, Satomi, M, Kimura, S 2001Sphingomonas cloacae sp. nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo. IntJ Syst Evol Microbiol51603610Google Scholar
  15. 15.
    Kästner, M, Breuer-Jammali, M, Mahro, B 1994Enumeration and characterisation of soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbonsAppl Environ Microbiol41267273Google Scholar
  16. 16.
    Keim, T, Francke, W, Schmidt, SK, Fortnagel, P 1999Catabolism of 2,7-dichloro-and 2,4,8-trichlorodibenzofuran by Sphingomonas sp. strain RW1J Ind Microbiol Biotechnol23359363Google Scholar
  17. 17.
    Khan, AA, Wang, RF, Cao, WW, Franklin, W, Cerniglia, CE 1996Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA–DNA hybridization, and 16S ribosomal DNA sequencingInt J Syst Bacteriol46466469Google Scholar
  18. 18.
    Kim, S, Chun, J, Bae, K, Kim, Y 2000Polyphasic assignment of an aromatic-degrading Pseudomonas sp., strain DJ77, in the genus Sphingomonas as Sphingomonas chungbukensis sp. nov. IntJ Syst Evol Microbiol5016411647Google Scholar
  19. 19.
    Kohler, HPE 1999Sphingomonas herbicidivorans MH: a versatile phenoxyalkanoic acid herbicide degraderJ Ind Microbiol Biotechnol23336340Google Scholar
  20. 20.
    Leys, NMEJ, Ryngaert, A, Bastiaens, L, Verstraete, EM, Top, W, Springael, D 2004Occurence and phylogenetic diversity of Sphingomonas in soils contaminated with polycyclic aromatic hydrocarbons (PAHs)Appl Environ Microbiol7019441955Google Scholar
  21. 21.
    Mergeay, M, Nies, D, Schlegel, HG, Gerits, J, Charles, P, Gijsegem, F 1985Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metalsJ Bacteriol162328334Google Scholar
  22. 22.
    Mohn, WW, Moore, ERB, Muttray, A 1999Lessons learned from Sphingomonas species that degrade abietane triterpenoidsJ Ind Microbiol Biotechnol23374379Google Scholar
  23. 23.
    Mueller, JG, Chapman, PJ, Blattmann, BO, Pritchard, PH 1990Isolation and characterisation of a fluoranthene-utilizing strains of Pseudomonas paucimobilisAppl Environ Microbiol5610791086Google Scholar
  24. 24.
    Mueller, JG, Devereux, R, Santavy, DL, Lantz, SE, Willis, SG, Pritchard, PH 1997Phylogenetic and physiological comparison of PAH-degrading bacteria from geographically diverse soilsAnt van Leeuwen71329343Google Scholar
  25. 25.
    Nagata, Y, Miyauchi, K, Takagi, M 1999Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26J Ind Microbiol Biotechnol23380390Google Scholar
  26. 26.
    Osterreicher-Ravid, D, Ron, EZ, Rosenberg, E 2000Horizontal transfer of an exopolymer complex from one bacterial species to anotherEnviron Microbiol2366372Google Scholar
  27. 27.
    Pinyakong, O, Habe, H, Supaka, N, Pinpanichkarn, P, Juntongjin, K, Yoshida, T, Furihata, K, Nojiri, H, Yamane, H, Omori, T 2000Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2FEMS Microbiol. Lett191115121Google Scholar
  28. 28.
    Pinyakong, O, Habe, H, Omori, T 2003The unique aromatic catabolic genes in sphingomonads degrading polycyclic aromatic hydrocarbons (PAHs)J Gen Appl Microbiol49119Google Scholar
  29. 29.
    Saitou, N, Nei, M 1987The neighbor-joining method: a new method for reconstructing phylogenetic treesMol Biol Evol4406425Google Scholar
  30. 30.
    Sorensen, SR, Ronen, Z, Aamand, J 2001Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturonAppl Environ Microbiol6754035409Google Scholar
  31. 31.
    Stolz, A 1999Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6J Ind Microbiol Biotechnol23391399Google Scholar
  32. 32.
    Stolz, A, Schmidt-Maag, C, Denner, E, Busse, H, Egli, T, Kampfer, P 2000Description of Sphingomonas xenophaga sp. nov. for strains BN6(T) and N,N which degrade xenobiotic aromatic compoundsInt J Syst Evol Microbiol503541Google Scholar
  33. 33.
    Story, SP, Parker, SH, Kline, JD, Tzeng, TR, Mueller, JG, Kline, EL 2000Identification of four structural genes and two putative promotors necessary for utilization of naphthalene, phenanthrene and fluoranthene by Sphingomonas paucimobilis var. EPA505Gene260155169Google Scholar
  34. 34.
    Story, SP, Parker, SH, Hayasaka, SS, Riley, MB, Kline, EL 2001Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene and phenanthrene by Sphingomonas paucimobilis var. EPA505J Ind Microbiol Biotechnol26369382Google Scholar
  35. 35.
    Takeuchi, M, Hamana, K, Hiraishi, A 2001Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analysesInt J Syst Evol Microbiol5114051417Google Scholar
  36. 36.
    Elsas, JD, Rosado, AS, Wolters, AC, Moore, E, Karlson, U 1998Quantitative detection of Sphingomonas chlorophenolica in soil via competitive polymerase chain reactionJ Appl Microbiol85463471Google Scholar
  37. 37.
    Vanbroekhoven, K, Ryngaert, A, Bastiaens, L, Vancanneyt, M, Wattiau, P, De Mot, R, Springael D (2004) Streptomycine as a selective agent to facilitate recovery and isolation of introduced and endogenous sphingomonads in environmental samples. Environ Microbiol 6: 1123–1136Google Scholar
  38. 38.
    Weissenfels, WD, Beyer, M, Klein, J, Rehm, H-J 1991Microbial metabolism of fluoranthene: isolation and identification of ring fission productsAppl Microbiol Biotechnol34528535Google Scholar
  39. 39.
    Willumsen, PA, Karlson, U 1998Effect of calcium on the surfactant tolerance of a fluoranthene degrading bacteriumBiodegradation9369379Google Scholar
  40. 40.
    Willumsen, PA, Karlson, U, Pritchard, PH 1998Response of fluoranthene-degrading bacteria to surfactantsAppl Microbiol Biotechnol50475483Google Scholar
  41. 41.
    Wittich, RM, Strompl, C, Moore, ERB, Blasco, R, Timmis, KN 1999Interaction of Sphingomonas and Pseudomonas in the degradation of chlorinated dibenzofuransJ Ind Microbiol Biotechnol23359363Google Scholar
  42. 42.
    Yrjala, K, Suomalainen, S, Suhonen, EL, Kilpi, S, Paulin, L, Romantschuk, M 1998Characterization and reclassification of an aromatic- and chloroaromatic-degrading Pseudomonas sp., strain HV36, as Sphingomonas sp. HV3Int J Syst Bacteriol4810571062Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • N.M. Leys
    • 1
    • 2
    • 3
  • A. Ryngaert
    • 1
  • L. Bastiaens
    • 1
  • E.M. Top
    • 2
    • 4
  • W. Verstraete
    • 2
  • D. Springael
    • 1
    • 5
    Email author
  1. 1.Environmental and Process TechnologyFlemish Institute for Technological Research (Vito)Belgium
  2. 2.Laboratory of Microbial Ecology and TechnologyUniversity of Ghent (UG)Belgium
  3. 3.Laboratory of MicrobiologyBelgian Nuclear Research Centre(SCK/CEN)BoeretangBelgium
  4. 4.Department of Biological SciencesUniversity of IdahoMoscowUSA
  5. 5.Laboratory of Soil and Water ManagementCatholic University of Leuven (KUL)Belgium

Personalised recommendations