, Volume 24, Issue 2, pp 279–293 | Cite as

Effect of carbon source during enrichment on BTEX degradation by anaerobic mixed bacterial cultures

  • Murthy Kasi
  • Tanush Wadhawan
  • John McEvoy
  • G. Padmanabhan
  • Eakalak Khan
Original Paper


A comprehensive study on the effects of different carbon sources during the bacterial enrichment on the removal performances of benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds when present as a mixture was conducted. Batch BTEX removal kinetic experiments were performed using cultures enriched with individual BTEX compounds or BTEX as a mixture or benzoate alone or benzoate–BTEX mixture. An integrated Monod-type non-linear model was developed and a ratio between maximum growth rate (μmax) and half saturation constant (Ks) was used to fit the non-linear model. A higher μmax/Ks indicates a higher affinity to degrade BTEX compounds. Complete removal of BTEX mixture was observed by all the enriched cultures; however, the removal rates for individual compounds varied. Degradation rate and the type of removal kinetics were found to be dependent on the type of carbon source during the enrichment. Cultures enriched on toluene and those enriched on BTEX mixture were found to have the greatest μmax/Ks and cultures enriched on benzoate had the least μmax/Ks. Removal performances of the cultures enriched on all different carbon sources, including the ones enriched on benzoate or benzoate–BTEX mixture were also improved during a second exposure to BTEX. A molecular analysis showed that after each exposure to the BTEX mixture, the cultures enriched on benzoate and those enriched on benzoate–BTEX mixture had increased similarities to the culture enriched on BTEX mixture.


Biodegradation Enrichment Mixed bacterial culture BTEX Anaerobic 


  1. Aburto-Medina A, Adetutu EM, Aleer S, Weber J, Patil SS, Sheppard PJ, Ball AS, Juhasz AL (2012) Comparison of indigenous and exogenous microbial populations during slurry phase biodegradation of long-term hydrocarbon-contaminated soil. Biodegradation. doi:10.1007/s10532-012-9563-8 PubMedGoogle Scholar
  2. Alvarez PJJ, Cronkhite LA, Hunt CS (1998) Use of benzoate to establish reactive buffer zones for enhanced attenuation of BTX migration: aquifer column experiments. Environ Sci Technol 32:509–515CrossRefGoogle Scholar
  3. Bielefeldt AR, Stensel HD (1999) Modeling competitive inhibition effects during biodegradation of BTEX mixtures. Water Res 33:707–714CrossRefGoogle Scholar
  4. Blackburn JW (1998) Bioremediation scaleup effectiveness: a Review. Bioremediat J 1:265–282CrossRefGoogle Scholar
  5. Boopathy R, Shields S, Nunna S (2012) Biodegradation of crude oil from the BP oil spill in the marsh sediments of Southeast Louisiana, USA. Appl Biochem Biotechnol. doi:10.1007/s12010-012-9603-1 PubMedGoogle Scholar
  6. Borden RC, Hunt MJ, Shafer MB, Barlaz MA (1997) Anaerobic biodegradation of BTEX in aquifer material. EPA/600/S-97/003Google Scholar
  7. Botton S, Parsons JR (2006) Degradation of BTEX compounds under iron-reducing conditions in contaminated aquifer microcosms. Environ Toxicol Chem 25:2630–2638PubMedCrossRefGoogle Scholar
  8. Burland SM, Edwards EA (1999) Anaerobic benzene biodegradation linked to nitrate reduction. Appl Environ Microbiol 65:529–533PubMedGoogle Scholar
  9. Chakraborty R, Coates JD (2004) Anaerobic degradation of monoaromatic hydrocarbons. Appl Microbiol Biotechnol 64:437–446PubMedCrossRefGoogle Scholar
  10. Chakraborty R, O’Connor SM, Chan E, Coates JD (2005) Anaerobic degradation of benzene, toluene, ethylbenzene and xylene by Dechloromonas strain RCB. Appl Environ Microbiol 71:8649–8655PubMedCrossRefGoogle Scholar
  11. Champion KM, Zengler K, Rabus R (1999) Anaerobic degradation of ethylbenzene and toluene in denitrifying strain EbN1 proceeds via independent substrate-induced pathways. J Mol Microbiol Biotechnol 1:157–164PubMedGoogle Scholar
  12. Coates JD, Chakraborty R, Lack JG, O’Connor SM, Cole KA, Bender KS, Achenbach LA (2001) Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas. Nature 411:1039–1043PubMedCrossRefGoogle Scholar
  13. Da Silva MLB, Alvarez PJJ (2004) Enhanced anaerobic biodegradation of benzene–toluene–ethylbenzene–xylene–ethanol mixtures in bioaugmented aquifer columns. Appl Environ Microbiol 70:4720–4726PubMedCrossRefGoogle Scholar
  14. Dou J, Liu X, Hu Z (2008) Substrate interactions during anaerobic biodegradation of BTEX by the mixed cultures under nitrate reducing conditions. J Hazard Mater 158:264–272PubMedCrossRefGoogle Scholar
  15. Evans PJ, Ling W, Goldschmidt B, Ritter ER, Young LY (1992) Metabolites formed during anaerobic transformation of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization. Appl Environ Microbiol 58:496–501PubMedGoogle Scholar
  16. Foght J (2008) Anaerobic biodegradation of aromatic hydrocarbons: pathways and prospects. J Mol Microbiol Biotechnol 15:93–120PubMedCrossRefGoogle Scholar
  17. Healey FP (1980) Slope of the Monod equation as an indicator of advantage in nutrient competition. Microb Ecol 5:281–286CrossRefGoogle Scholar
  18. Hunt MJ, Borden RC, Barlaz MA (1998) Determining anaerobic BTEX decay rates in a contaminated aquifer. J Hydrol Eng 3:285–293CrossRefGoogle Scholar
  19. Hutchins SR, Sewell GW, Kovacs DA, Smith GA (1991) Biodegradation of aromatic hydrocarbons by aquifer microorganisms under denitrifying conditions. Environ Sci Technol 25:68–76CrossRefGoogle Scholar
  20. Kasi M, McEvoy J, Padmanabhan G, Khan E (2011) Groundwater remediation using enricher reactor—permeable reactive biobarrier for periodically absent contaminants. Water Environ Res 83:603–612PubMedCrossRefGoogle Scholar
  21. Kniemeyer O, Heider J (2001) (S)-1-Phenylethanol dehydrogenase of Azoarcus sp. strain EbN1, an enzyme of anaerobic ethylbenzene catabolism. Arch Microbiol 176:129–135PubMedCrossRefGoogle Scholar
  22. Krieger CJ, Beller HR, Reinhard M, Spormann AM (1999) Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp. strain T. J Bacteriol 181:6403–6410PubMedGoogle Scholar
  23. Leutwein C, Heider J (1999) Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate (R)-(+)-benzylsuccinate. Microbiol 145:3265–3271Google Scholar
  24. Li G, Huang DN, Lerner DN, Zhang X (2000) Enrichment of degrading microbes and bioremediation of petrochemical contaminants in polluted soil. Water Res 34:3845–3853CrossRefGoogle Scholar
  25. Margesin R, Schinner F (2001) Biodegradation and bioremediation of hydrocarbons in extreme environments. Appl Microbiol Biotechnol 56:650–663PubMedCrossRefGoogle Scholar
  26. Maxwell CR, Baqai HA (1995) Remediation of petroleum hydrocarbons by inoculation with laboratory-cultured microorganisms. In: Hinchee RE, Fredrickson J, Alleman BC (eds) Bioaugmentation for site remediation. Battelle Press, Columbus, pp 129–137Google Scholar
  27. Nales M, Butler B, Edwards E (1998) Anaerobic benzene biodegradation: a microcosm survey. Bioremed J 2:125–144CrossRefGoogle Scholar
  28. Olaniran AO, Pillay D, Pillay B (2006) Biostimulation and bioaugmentation enhances aerobic biodegradation of dichloroethenes. Chemosphere 63:600–608PubMedCrossRefGoogle Scholar
  29. Otte M-P, Gagnon J, Comeau Y, Matte N, Greet CW, Samson R (1994) Activation of an indigenous microbial consortium for bioaugmentation of pentachlorophenol/creosote contaminated soils. Appl Microbiol Biotechnol 40:926–932CrossRefGoogle Scholar
  30. Patterson BM, Pribac F, Barber C, Davis GB, Gibbs R (1993) Biodegradation and retardation of PCE and BTEX compounds in aquifer material from Western Australia using large-scale columns. J Contam Hydrol 14:261–278CrossRefGoogle Scholar
  31. Payne WJ (1973) Reduction of nitrogenous oxides by microorganisms. Bact Rev 37:409–452PubMedGoogle Scholar
  32. Portier R, Bianchini M, Fujisaki K, Henry C, McMilin D (1988) Comparison of effective toxicant biotransformation by autochthonous microorganisms and commercially available cultures in the in situ reclamation of abandoned industrial sites. Schriftenr Ver Wasser Boden Lufthyg 80:273–292Google Scholar
  33. Pritchard PH, Mueller JG, Rogers JC, Kremer FV, Glaser JA (1992) Oil spill bioremediation: experiences, lessons and results from the exxon valdez oil spill in Alaska. Biodegradation 3:315–335CrossRefGoogle Scholar
  34. Rabus R, Heider J (1998) Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria. Arch Microbiol 170:377–384CrossRefGoogle Scholar
  35. Shen W, Chen H, Pan S (2008) Anaerobic biodegradation of 1,4-dioxane by sludge enriched with iron-reducing microorganisms. Bioresource Technol 99:2483–2487CrossRefGoogle Scholar
  36. Stallwood B, Shears J, Williams PA, Hughes KA (2005) Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica. J Appl Microbiol 99:794–802PubMedCrossRefGoogle Scholar
  37. Suarez MP, Rifai HS (1999) Biodegradation rates for fuel hydrocarbons and chlorinated solvents in groundwater. Bioremediat J 3:337–362CrossRefGoogle Scholar
  38. Tellez GT, Nirmalakhandan N, Gardea-Torresdey JL (2002) Performance evaluation of an activated sludge system for removing petroleum hydrocarbons from oilfield produced water. Adv Environ Res 6:455–470CrossRefGoogle Scholar
  39. Triguerosa DEG, Módenesa AN, Kroumov AD, Espinoza-Quiñonesa FR (2010) Modeling of biodegradation process of BTEX compounds: kinetic parameters estimation by using particle swarm global optimizer. Process Biochem 45:1355–1361CrossRefGoogle Scholar
  40. Tyagi M, da Fonseca M, de Carvalho MR (2011) Bioaugmentation and biostimulation strategies to improve the effectiveness of bioremediation processes review paper. Biodegradation 22:231–241PubMedCrossRefGoogle Scholar
  41. Van der Zaan BM, Saia FT, Stams AJM, Plugge CM, de Vos WM, Smidt H, Langenhoff AAM, Gerritse J (2012) Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process. Environ Microbiol. doi:10.1111/j.1462-2920.2012.02697.x PubMedGoogle Scholar
  42. Wadhawan T, McEvoy JM, Prüß BM, Khan E (2010) Assessing tetrazolium and ATP assays for rapid in situ viability quantification of bacterial cells entrapped in hydrogel beads. Enzyme Microb Tech 47:166–173CrossRefGoogle Scholar
  43. Weelink AAB, van Eekert HA, Stams AJM (2010) Degradation of BTEX by anaerobic bacteria: physiology and application. Rev Environ Sci Biotechnol 9:359–385Google Scholar
  44. Weiner JM, Lovley DR (1998) Anaerobic benzene degradation in petroleum-contaminated aquifer sediments after inoculation with a benzene-degrading enrichment. Appl Environ Microbiol 64:775–778PubMedGoogle Scholar
  45. Widdel F, Rabus R (2001) Anaerobic biodegradation of saturated and aromatic hydrocarbons. Curr Opin Biotechnol 12:259–276PubMedCrossRefGoogle Scholar
  46. Yoon H, Klinzing G, Blanch HW (1977) Competition for mixed substrates by microbial populations. Biotechnol Bioeng 19:1193–1210PubMedCrossRefGoogle Scholar
  47. Zhao X, Wang Y, Ye Z, Borthwick AGL, Ni J (2006) Oil field wastewater treatment in biological aerated filter by immobilized microorganisms. Process Biochem 41:1475–1483CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Murthy Kasi
    • 1
    • 2
  • Tanush Wadhawan
    • 1
  • John McEvoy
    • 3
  • G. Padmanabhan
    • 1
  • Eakalak Khan
    • 1
  1. 1.Department of Civil EngineeringNorth Dakota State UniversityFargoUSA
  2. 2.Moore Engineering, Inc.West FargoUSA
  3. 3.Department of Veterinary and Microbiological SciencesNorth Dakota State UniversityFargoUSA

Personalised recommendations