, Volume 22, Issue 5, pp 973–982 | Cite as

Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing

  • Paula M. Martínez-Lavanchy
  • Anja Bettina Dohrmann
  • Gwenaël Imfeld
  • Karin Trescher
  • Christoph C. Tebbe
  • Hans-Hermann Richnow
  • Ivonne NijenhuisEmail author
Original Paper


Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of 13C-labelled MCB demonstrated that the community produced 13CO2 as a metabolite at slightly increasing rates over a period of 1,051 days while no 13C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.


Biodegradation Contamination Chlorobenzenes Bitterfeld Stable isotope probing 



We thank Heidrun Paschke (UFZ Dept. Groundwater Remediation) for providing geochemical data. P. M. M-L. was funded by a European Union Marie Curie Early Stage Training Fellowship (contract number MEST-CT-2004-8332). This work was partially supported by a collaborative project (BACSIN, Contract No. 211684) from the European Commission within its Seventh Framework Program. The project was financially supported by the Helmholtz Centre for Environmental Research.


  1. Adrian L, Gorisch H (2002) Microbial transformation of chlorinated benzenes under anaerobic conditions. Res Microbiol 153:131–137PubMedCrossRefGoogle Scholar
  2. Agteren MHv, Keuning S, Janssen DB (1998) Handbook on biodegradation and biological treatment of hazardous organic compounds. Kluwer Academic Publishers, Dordrecht, pp 363–378Google Scholar
  3. Alfreider A, Vogt C, Babel W (2002) Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis. Syst Appl Microbiol 25:232–240PubMedCrossRefGoogle Scholar
  4. Anderson RT, Rooney-Varga JN, Gaw CV, Lovley DR (1998) Anaerobic benzene oxidation in the Fe(III) reduction zone of petroleum contaminated aquifers. Environ Sci Technol 32:1222–1229CrossRefGoogle Scholar
  5. Braeckevelt M et al (2007) Biodegradation of chlorobenzene in a constructed wetland treating contaminated groundwater. Water Sci Technol 56:57–62PubMedGoogle Scholar
  6. Coates JD, Chakraborty R, McInerney MJ (2002) Anaerobic benzene biodegradation—a new era. Res Microbiol 153:621–628PubMedCrossRefGoogle Scholar
  7. Dohrmann AB, Tebbe CC (2005) Effect of elevated tropospheric ozone on the structure of bacterial communities inhabiting the rhizosphere of herbaceous plants native to Germany. Appl Environ Microbiol 71:7750–7758PubMedCrossRefGoogle Scholar
  8. Edwards EA, Grbić-Galić D (1992) Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions. Appl Environ Microbiol 58:2663–2666PubMedGoogle Scholar
  9. Fahy A, McGenity TJ, Timmis KN, Ball AS (2006) Heterogeneous aerobic benzene-degrading communities in oxygen-depleted groundwaters. FEMS Microbiol Ecol 58:260–270PubMedCrossRefGoogle Scholar
  10. Fung JM, Weisenstein BP, Mack EE, Vidumsky JE, Ei TA, Zinder SH (2009) Reductive dehalogenation of dichlorobenzenes and monochlorobenzene to benzene in microcosms. Environ Sci Technol 43:2302–2307PubMedCrossRefGoogle Scholar
  11. Harris JK, Kelley ST, Pace NR (2004) New perspective on uncultured bacterial phylogenetic division OP11. Appl Environ Microbiol 70:845–849PubMedCrossRefGoogle Scholar
  12. Janssen PH, Schuhmann A, Bak F, Liesack W (1996) Disproportionation of inorganic sulfur compounds by the sulfate-reducing bacterium Desulfocapsai thiozymogenes gen. nov., sp. nov. Arch Microbiol 166:184–192CrossRefGoogle Scholar
  13. Kasai Y, Takahata Y, Manefield M, Watanabe K (2006) RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater. Appl Environ Microbiol 72:3586–3592PubMedCrossRefGoogle Scholar
  14. Kaschl A et al (2005) Isotopic fractionation indicates anaerobic monochlorobenzene biodegradation. Environ Toxicol Chem 24:1315–1324PubMedCrossRefGoogle Scholar
  15. Kodama Y, Watanabe K (2004) Sulfuricurvum kujiense gen. nov., sp nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity. Int J Syst Evol Microbiol 54:2297–2300PubMedCrossRefGoogle Scholar
  16. Kunapuli U, Lueders T, Meckenstock RU (2007) The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation. ISME J 1:643–653PubMedCrossRefGoogle Scholar
  17. Lovley DR (2000) Anaerobic benzene degradation. Biodegradation 11:107–116PubMedCrossRefGoogle Scholar
  18. Lueders T, Manefield M, Friedrich MW (2004) Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ Microbiol 6:73–78PubMedCrossRefGoogle Scholar
  19. Manefield M, Whiteley AS, Ostle N, Ineson P, Bailey MJ (2002) Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function. Rapid Commun Mass Spectrom 16:2179–2183PubMedCrossRefGoogle Scholar
  20. Miyake D et al (2007) Thiosulfate oxidation by a moderately thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus. Arch Microbiol 188:199–204PubMedCrossRefGoogle Scholar
  21. Musat F, Widdel F (2008) Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype. Environ Microbiol 10:10–19PubMedGoogle Scholar
  22. Neufeld JD et al (2007a) DNA stable-isotope probing. Nat Protoc 2:860–866PubMedCrossRefGoogle Scholar
  23. Neufeld JD, Wagner M, Murrell JC (2007b) Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J 1:103–110PubMedCrossRefGoogle Scholar
  24. Nijenhuis I, Stelzer N, Kastner M, Richnow HH (2007) Sensitive detection of anaerobic monochlorobenzene degradation using stable isotope tracers. Environ Sci Technol 41:3836–3842PubMedCrossRefGoogle Scholar
  25. Nowak J, Kirsch NH, Hegemann W, Stan HJ (1996) Total reductive dechlorination of chlorobenzenes to benzene by a methanogenic mixed culture enriched from Saale river sediment. Appl Microbiol Biotechnol 45:700–709CrossRefGoogle Scholar
  26. Oka AR, Phelps CD, McGuinness LM, Mumford A, Young LY, Kerkhof LJ (2008) Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing. Appl Environ Microbiol 74:6476–6480PubMedCrossRefGoogle Scholar
  27. Phelps CD, Zhang X, Young LY (2001) Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium. Environ Microbiol 3:600–603PubMedCrossRefGoogle Scholar
  28. Radajewski S, McDonald IR, Murrell JC (2003) Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Curr Opin Biotechnol 14:296–302PubMedCrossRefGoogle Scholar
  29. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  30. Schmalenberger A, Hodge S, Bryant A, Hawkesford MJ, Singh BK, Kertesz MA (2008) The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial wheat rhizosphere communities exposed to different sulfur fertilization regimes. Environ Microbiol 10:1486–1500PubMedCrossRefGoogle Scholar
  31. Schwieger F, Tebbe CC (1998) A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl Environ Microbiol 64:4870–4876PubMedGoogle Scholar
  32. Stelzer N et al (2009) Integrative approach to delineate natural attenuation of chlorinated benzenes in anoxic aquifers. Environ Pollut 157:1800–1806PubMedCrossRefGoogle Scholar
  33. Sun W et al (2009) Molecular characterization and in situ quantification of anoxic arsenite-oxidizing denitrifying enrichment cultures. FEMS Microbiol Ecol 68:72–85PubMedCrossRefGoogle Scholar
  34. Vogt C, Simon D, Alfreider A, Babel W (2004) Microbial degradation of chlorobenzene under oxygen-limited conditions leads to accumulation of 3-chlorocatechol. Environ Toxicol Chem 23:265–270PubMedCrossRefGoogle Scholar
  35. Weelink SA et al (2008) Isolation and characterization of Alicycliphilus denitrificans strain BC, which grows on benzene with chlorate as the electron acceptor. Appl Environ Microbiol 74:6672–6681PubMedCrossRefGoogle Scholar
  36. Whitby C et al (2005) Stable isotope probing links taxonomy with function in microbial communities. ASM News 71:169–173Google Scholar
  37. Whiteley AS, Manefield M, Lueders T (2006) Unlocking the ‘microbial black box’ using RNA-based stable isotope probing technologies. Curr Opin Biotechnol 17:67–71PubMedCrossRefGoogle Scholar
  38. Yagi JM, Sims D, Brettin T, Bruce D, Madsen EL (2009) The genome of Polaromonas naphthalenivorans strain CJ2, isolated from coal tar-contaminated sediment, reveals physiological and metabolic versatility and evolution through extensive horizontal gene transfer. Environ Microbiol 11:2253–2270PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Paula M. Martínez-Lavanchy
    • 1
  • Anja Bettina Dohrmann
    • 3
  • Gwenaël Imfeld
    • 4
  • Karin Trescher
    • 3
  • Christoph C. Tebbe
    • 3
  • Hans-Hermann Richnow
    • 2
  • Ivonne Nijenhuis
    • 2
    Email author
  1. 1.Department of Environmental BiotechnologyHelmholtz Centre for Environmental Research, UFZLeipzigGermany
  2. 2.Department of Isotope BiogeochemistryHelmholtz Centre for Environmental Research, UFZLeipzigGermany
  3. 3.Institut für BiodiversitätJohann Heinrich von Thünen-Institut (vTI), Bundesforschungsinstitut für Ländliche Räume, Wald und FischereiBraunschweigGermany
  4. 4.Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS)Université de Strasbourg/ENGEES, CNRSStrasbourgFrance

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