The Use of Multiple Lines of Evidence to Substantiate Anaerobic BTEX Degradation in Groundwater

  • Janneke WittebolEmail author
  • Inez DinklaEmail author
Part of the Springer Protocols Handbooks book series (SPH)


Aromatic compounds are nowadays still of major environmental concern. These compounds have been proven to be biodegradable under both aerobic and anaerobic conditions. Under anaerobic conditions several biodegradation pathways are proposed, but the bacteria and specific genes involved remain largely unknown. The detection of the actual biological degradation potential and expected kinetics of degradation in the field are therefore a challenge. Usually, a combination of different lines of evidence is used to determine and predict the biodegradation of BTEX under anaerobic conditions in the field. These include compound-specific monitoring of pollutants and intermediates in groundwater, laboratory degradation tests, stable isotope probing and application of BACTRAPs and/or microcosms. Each of these methods provides part but indirect evidence for the actual in situ biodegradation kinetics. Molecular monitoring of biodegradation of aromatic compounds in the field is not commonly used but can provide important additional evidence, especially when directed to target RNA.

Molecular analysis of functional genes involved, in combination with other lines of evidence, can be used to directly and accurately determine the degradation potential. The protocol described in this chapter allows for the accurate assessment of the BTEX biodegradation potential on-site following four steps:
  1. 1)

    Groundwater sampling using conventional, dialysis or microcosm sampling

  2. 2)

    Groundwater characterisation

  3. 3)

    Sampling protocol for molecular analyses

  4. 4)

    Molecular analyses of functional genes



Anaerobic BTEX degradation Aromatic compounds Bioremediation Extra line of evidence Functional genes Molecular tool qPCR Site characterisation 


  1. 1.
    Lovley D (2001) Bioremediation - anaerobes to the rescue. Science 293:1444–1446CrossRefPubMedGoogle Scholar
  2. 2.
    Harwood C, Burchhardt G, Hermann H, Georg F (1999) Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway. FEMS Microbiol Rev 22(5):439–458CrossRefGoogle Scholar
  3. 3.
    Lueders T, Von Netzer F (2014) Functional genes for anaerobic hydrocarbon degrading microbes. In: McGenity TJ et al (eds) Hydrocarbon and lipid microbiology protocols, Springer protocols handbooks. Springer, BerlinGoogle Scholar
  4. 4.
    Kleinsteuber S, Schleinitz KM, Vogt C (2012) Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers. Appl Microbiol Biotechnol 94:851–873CrossRefPubMedGoogle Scholar
  5. 5.
    Kühner S, Wöhlbrand L, Fritz I, Wruck W, Hultschig C, Hufnagel P, Kube M, Reinhardt R, Rabus R (2005) Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1. J Bacteriol 187(4):1493–1503CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Weelink SAB, van Eekert MHA, Stams AJM (2010) Degradation of BTEX by anaerobic bacteria: physiology and application. Rev Environ Sci Biotechnol 9:359–385CrossRefGoogle Scholar
  7. 7.
    Selesi D, Jehmlich N, von Bergen M, Schmidt F, Rattei T, Tischler P, Lueders T, Meckenstock RU (2010) Combined genomic and proteomic approaches identify gene clusters involved in anaerobic 2-methylnaphthalene degradation in the sulfate-reducing enrichment culture N47. J Bacteriol 192(1):295–306CrossRefPubMedGoogle Scholar
  8. 8.
    van der Zaan B, Saia FT, Stams AJ, Plugge CM, de Vos WM, Smidt H, Langenhoff AA, Gerritse J (2012) Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process. Environ Microbiol 14:1171–1181CrossRefPubMedGoogle Scholar
  9. 9.
    Lovley D, Coates JD, Woodward JC, Phillips E (1995) Benzene oxidation coupled to sulfate reduction. Appl Environ Microbiol 61(2):953–958PubMedPubMedCentralGoogle Scholar
  10. 10.
    Fuchs G, Boll M, Heider J (2011) Microbial degradation of aromatic compounds—from one strategy to four. Nat Rev Microbiol 9:803–816CrossRefPubMedGoogle Scholar
  11. 11.
    Noguchi M, Kurisu F, Kasuga I, Furumai H (2014) Time-resolved DNA stable isotope probing links Desulfobacterales- and Coriobacteriaceae-related bacteria to anaerobic degradation of benzene under methanogenic conditions. Microbes Environ 29(2):191–199CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Radajewski S, Ineson P, Parekh NR, Murrell JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649CrossRefPubMedGoogle Scholar
  13. 13.
    EPA U (1999) Use of monitored natural attenuation at superfund, RCRA corrective action, and underground storage tank sites. United States Environmental Protection Agency, WashingtonGoogle Scholar
  14. 14.
    E. Commission (2010) Strategies against chemical pollution of surface waters, 2009/90/EC. Available: Accessed 27 8 2015
  15. 15.
    Röling WF, van Breukelen BM, Braster M, Lin B, van Verseveld HW (2001) Relationships between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer. Appl Environ Microbiol 67(10):4619–4629CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Weiss J, Cozzarelli O (2008) Biodegradation in contaminated aquifers: incorporating microbial/molecular methods. Ground Water 46(2):305–322CrossRefPubMedGoogle Scholar
  17. 17.
    Aburto-Medina A, Ball A (2015) Microorganisms involved in anaerobic benzene degradation. Ann Microbiol 65:1201–1213CrossRefGoogle Scholar
  18. 18.
    Langenhoff A and van Ras N (2006) Anaërobe afbraak van benzeen, het ultieme bewijs (PT04-120) SKBGoogle Scholar
  19. 19.
    Fischer A, Theuerkorn K, Stelzer N, Gehre M, Thullner M, Richnow H (2007) Applicability of stable isotope fractionation analysis for the characterization of benzene biodegradation in a BTEX-contaminated aquifer. Environ Sci Technol 41:3689–3696CrossRefPubMedGoogle Scholar
  20. 20.
    Fischer A, Gehre M, Breitfeld J, Richnow H, Vogt C (2009) Carbon and hydrogen isotope fractionation of benzene during biodegradation under sulfate-reducing conditions: a laboratory to field site approach. Rapid Commun Mass Spectrom 23:2439–2447CrossRefPubMedGoogle Scholar
  21. 21.
    Vogt C, Kleinsteuber S, Richnow H (2011) Anaerobic benzene degradation by bacteria. Microb Biotechnol 4:710–724CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Van Ras N, Winters R, Lieten S, Dijkhuis J, Henssen M (2007) Sustainability of natural attenuation of aromatics (BTEX). NICOLE, Appeldoorn, NetherlandsGoogle Scholar
  23. 23.
    Liang X, Devine CE, Nelson J, Sherwood LB, Zinder S, Edwards E (2013) Anaerobic Conversion of Chlorobenzene and Benzene to CH4 and CO2 in Bioaugmented Microcosms. Environ Sci Technol 47(5):2378–2385CrossRefPubMedGoogle Scholar
  24. 24.
    Herrmann S, Kleinsteuber S, Neu TR, Richnow HH, Vogt C (2008) Enrichment of anaerobic benzene-degrading microorganisms by in situ microcosms. FEMS Microbiol Ecol 63:94–106CrossRefPubMedGoogle Scholar
  25. 25.
    Luitwieler M (2012) Assessment of in situ biodegradation of benzene in a contaminated aquifer using BACTRAPs (in Dutch, confidential), 20114102/8647. Bioclear bvGoogle Scholar
  26. 26.
    Schuring C, Melo VA, Miltner A, Kaestner M (2014) Characterisation of microbial activity in the framework of natural attenuation without groundwater monitoring well?: an new Direct-Push probe. Environ Sci Pollut Res Int 21:9002–9015CrossRefGoogle Scholar
  27. 27.
    Hendrickx B, Junca H, Vosahlova J, Lindner A, Rüegg I, Bucheli-Witschel M, Faber F, Egli T, Mau M, Schlömann M, Brennerova M, Brenner V, Pieper DH, Top EM, Dejonghe W, Bastiaens L, Springael D (2006) Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site. J Microbiol Methods 64(2):250–265CrossRefPubMedGoogle Scholar
  28. 28.
    Holm PE, Nielsen PH, Albrechtsen HJ, Christensen TH (1992) Importance of unattached bacteria and bacteria attached to sediment in determining potentials for degradation of xenobiotic organic contaminants in an aerobic aquifer. Appl Environ Microbiol 58:3020–3026PubMedPubMedCentralGoogle Scholar
  29. 29.
    Lieten S, van Ras N, Wittebol J (2013) Biodegradation capacity in Utrecht – using innovative next level technologies. Citychlor, UtrechtGoogle Scholar
  30. 30.
    Krooneman J, Geurkink B, Zandstra JM, van der Waarde JJ, Smittenberg J, Westerman I, Röling W, Braster M, Bin L, Feis B, van Verseveld H, Akkermans A, Ramirez-Saad H, Smidt H, Dekker B, de Vries M, van Duuren J and Pedro T (2000) Microbiological characterization of contaminated soil and groundwater, NOBIS Dutch research programme biotechnology in-situ remediation, September 2000Google Scholar
  31. 31.
    Dijkhuis JE, Henssen M (2003) Sustainability of biodegradation, no longer a matter of mother Chicken reading the tea leafs! A new methodology on biodegradation performance (in Dutch). 5:186–189.
  32. 32.
    Van Bemmel M, Werf AWvd, Henssen MJC and Ras NJPv (2007) Experiences with bioaugmentation in full-scale applications. In: Proceedings of the ninth international in situ and on-site bioremediation symposium, BaltimoreGoogle Scholar
  33. 33.
    Maphosa F, Lieten SH, Dinkla I, Stams AJ, Smidt H, Fennell DE (2012) Ecogenomics of microbial communities in bioremediation of chlorinated contaminated sites. Front Microbiol 3:351CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Scholz M, Lo C-C, Chain P (2012) Next generation sequencing and bioinformatic bottlenecks: the current state of metagenomic data analysis. Curr Opin Biotechnol 23:9–15CrossRefPubMedGoogle Scholar
  35. 35.
    S. I. K. B. (SIKB) (2007) Available: Accessed 1 Dec 2014

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Bioclear bvEnvironmental ConsultancyGroningenThe Netherlands
  2. 2.Soil & WaterBioclear BVGroningenThe Netherlands

Personalised recommendations