, Volume 20, Issue 3, pp 419–431 | Cite as

Benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate

  • Margarete Bucheli-Witschel
  • Tina Hafner
  • Irene Rüegg
  • Thomas Egli
Original Paper


The regulation of benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate was investigated in batch and continuous culture. In batch culture, R. pickettii PKO1 achieved a maximum specific growth rate with benzene of 0.18 h−1, while succinate allowed much faster growth (μmax = 0.5 h−1). Under carbon excess conditions succinate repressed benzene consumption resulting in diauxic growth whereas under carbon-limited conditions in the chemostat both substrates were used simultaneously. Moreover, the effect of succinate on the adaptation towards growth with benzene was investigated in carbon-limited continuous culture at a dilution rate of 0.1 h−1 by changing the inflowing carbon substrate from succinate to different mixtures of benzene and succinate. The adaptation process towards utilisation of benzene was rather complex. Three to seven hours after the medium shift biomass production from benzene started. Higher proportions of succinate in the mixture had a positive effect on both the onset of biomass production and on the time required for induction of benzene utilisation. Strikingly, after the initial increase in biomass and benzene-catabolising activities, the culture collapsed regularly and wash-out of biomass was observed. After a transient phase of low biomass concentrations growth on benzene resumed so that finally rather stable and high biomass concentrations were reached. The decrease in biomass and degradative activities cannot be explained so far, but the possibilities of either intoxication of the cells by benzene itself, or of inhibition by degradation intermediates were ruled out.


Benzene Continuous culture Adaptation process Ralstonia pickettii PKO1 Succinate 



Benzene, toluene, ethylbenzene and xylenes


Optical density


Dissolved organic carbon




3-Phenylpropionic acid



This work was performed within the scope of the EU project QLK3-CT-2000-00731 and financially supported by grant BBW-Nr. 99.0821 from the Swiss Federal Office for Education and Science.


  1. Alagappan G, Cowan R (2003) Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures. Biotechnol Bioeng 83:798–809. doi: 10.1002/bit.10729 PubMedCrossRefGoogle Scholar
  2. Bally M, Egli T (1996) Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in carbon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl Environ Microbiol 62:133–140PubMedGoogle Scholar
  3. Baumann B, Snozzi M, Zehnder AJB, van der Meer JR (1996) Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes. J Bacteriol 178:4367–4374PubMedGoogle Scholar
  4. Bordel S, Munoz R, Diaz LF, Villaverde S (2007) New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites. Appl Microbiol Biotechnol 74:857–866. doi: 10.1007/s00253-006-0724-8 PubMedCrossRefGoogle Scholar
  5. Bringmann G, Kühn R (1980) Comparison of the toxicity thresholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test. Water Res 14:231–241. doi: 10.1016/0043-1354(80)90093-7 CrossRefGoogle Scholar
  6. Bruins MR, Kapil S, Oehme FW (2000) Pseudomonas pickettii: a common soil and aerobic groundwater bacteria with pathogenic and biodegradation properties. Ecotoxicol Environ Saf 47:105–111. doi: 10.1006/eesa.2000.1951 PubMedCrossRefGoogle Scholar
  7. Byrne AM, Olsen RH (1996) Cascade regulation of the toluene-3-monooxygenase operon (tbuA1UBVA2C) of Burkholderia pickettii PKO1: role of the tbuA1 promotor (PtbuA1) in the expression of its cognate activator TbuT. J Bacteriol 178:6327–6337PubMedGoogle Scholar
  8. Byrne AM, Kukor JJ, Olsen RH (1995) Sequence analysis of the gene cluster encoding toluene-3-monooxygenase from Pseudomonas pickettii PKO1. Gene 154:65–70. doi: 10.1016/0378-1119(94)00844-I PubMedCrossRefGoogle Scholar
  9. del Castillo T, Ramos JL (2007) Simultaneous catabolite repression between glucose and toluene metabolism in Pseudomonas putida is channeled through different signal pathways. J Bacteriol 189:6602–6610. doi: 10.1128/JB.00679-07 PubMedCrossRefGoogle Scholar
  10. Duetz WA, van Andel JG (1991) Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture. J Gen Microbiol 137:1369–1374PubMedGoogle Scholar
  11. Egli T (1995) The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. In: Jones GN (ed) Advances in microbial ecology. Plenum, New York, pp 305–386Google Scholar
  12. Egli T (2002) Microbial degradation of pollutants at low concentrations and in the presence of alternative carbon substrates: emerging patterns. In: Agathos SN, Reineke W (eds) Biotechnology for the environment: strategy and fundamentals. Kluwer, Dordrecht, pp 131–139Google Scholar
  13. Egli T, Weilenmann HU, El-Banna T, Auling G (1988) Gram-negative, aerobic, nitrilotriacetate-utilizing bacteria from wastewater and soil. Syst Appl Microbiol 10:297–305Google Scholar
  14. Evans CGT, Herbert D, Tempest DW (1970) The continuous cultivation of microorganisms—construction of a chemostat. In: Norris JR, Ribbons DW (eds) Methods in microbiology (2). Academic Press, London, pp 277–327Google Scholar
  15. Fang J, Barvelon MJ, Alvarez PJJ (2000) Phospholipid compositional changes of five pseudomonas archetypes grown with and without toluene. Appl Microbiol Biotechnol 54:382–389. doi: 10.1007/s002530000389 PubMedCrossRefGoogle Scholar
  16. Finette BA, Gibson DT (1988) Initial studies on the regulation of toluene degradation by Pseudomonas putida F1. Biocatalysis 2:29–37. doi: 10.3109/10242428808998177 CrossRefGoogle Scholar
  17. Gulensoy N, Alvarez PJ (1999) Diversity and correlation of specific aromatic hydrocarbon biodegradation capabilities. Biodegradation 10:331–340. doi: 10.1023/A:1008318405882 PubMedCrossRefGoogle Scholar
  18. Heiss G, Stolz A, Kuhm AE, Müller C, Klein J, Altenbuchner J, Knackmuss H-J (1995) Characterization of a 2, 3-dihydroxybiphenyl dioxygenase from the naphtalenesulfonate-degrading bacterium strain BN6. J Bacteriol 177:5865–5871PubMedGoogle Scholar
  19. Isken S, Derks A, Wolffs PF, de Bont JA (1999) Effect of organic solvents on the yield of solvent-tolerant Pseudomonas putida S12. Appl Environ Microbiol 65:2631–2635PubMedGoogle Scholar
  20. Jindrova E, Chocova M, Demnerova K, Brenner V (2002) Bacterial aerobic degradation of benzene toluene, ethylbenzene and xylene. Folia Microbiol (Praha) 47:83–93. doi: 10.1007/BF02817664 CrossRefGoogle Scholar
  21. Johnson DR, Park J, Kukor JJ, Abriola LM (2006) Effect of carbon starvation on toluene degradation activity by toluene monooxygenase-expressing bacteria. Biodegradation 17:437–445. doi: 10.1007/s10532-005-9014-x PubMedCrossRefGoogle Scholar
  22. Kahng H-Y, Byrne AM, Olsen RH, Kukor JJ (2000) Characterization and role of tbuX in utilization of toluene by Ralstonia pickettii PKO1. J Bacteriol 182:1232–1242. doi: 10.1128/JB.182.5.1232-1242.2000 PubMedCrossRefGoogle Scholar
  23. Kim H, Jaffé PR (2007) Spatial distribution and physiological state of bacteria in a sand column experiment during the biodegradation of toluene. Water Res 41:2089–2100. doi: 10.1016/j.watres.2007.02.018 PubMedCrossRefGoogle Scholar
  24. Kovarova K, Käch A, Chaloupka V, Egli T (1997) Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: dynamics of growth and substrate consumption. Biodegradation 7:2619–2624Google Scholar
  25. Kukor JJ, Olsen RH (1990) Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1c. J Bacteriol 172:4624–4630PubMedGoogle Scholar
  26. Kukor JJ, Olsen RH (1991) Genetic organization and regulation of a meta-cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1. J Bacteriol 173:4587–4594PubMedGoogle Scholar
  27. Kukor JJ, Olsen RH (1992) Complete nucleotide sequence of tbuD, the gene encoding phenol/cresol hydroxylase from Pseudomonas pickettii PKO1, and functional analysis of the encoded enzyme. J Bacteriol 174:6518–6526PubMedGoogle Scholar
  28. Kukor JJ, Olsen RH (1996) Catechol 2, 3-dioxygenases functional in oxygen-limited (hypoxic) environments. Appl Environ Microbiol 62:1728–1740PubMedGoogle Scholar
  29. Leahy JG, Olsen RH (1997) Kinetics of toluene degradation by toluene-oxidizing bacteria as a function of oxygen concentration, and the effect of nitrate. FEMS Microbiol Ecol 23:23–30. doi: 10.1111/j.1574-6941.1997.tb00387.x CrossRefGoogle Scholar
  30. Leddy MB, Phipps DW, Ridgway HF (1995) Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida. J Bacteriol 177:4713–4720PubMedGoogle Scholar
  31. Leveau JHJ, König F, Füchslin HP, Werlen C, van der Meer JR (1999) Dynamics of multigene expression during catabolic adaptation of Ralstonia eutropha JMP134 (pJP4) to the herbicide 2, 4-dichlorophenoxyacetate. Mol Microbiol 33:396–406. doi: 10.1046/j.1365-2958.1999.01483.x PubMedCrossRefGoogle Scholar
  32. Lovley DR (2000) Anaerobic benzene degradation. Biodegradation 11:107–116. doi: 10.1023/A:1011191220463 PubMedCrossRefGoogle Scholar
  33. Massol-Deya A, Weller R, Rios-Hernandez L, Zhou JZ, Hickey RF, Tiedje JM (1997) Succession and convergence of biofilm communities in fixed-film reactors treating aromatic hydrocarbons in groundwater. Appl Environ Microbiol 63:270–276PubMedGoogle Scholar
  34. Morita RY (1993) Bioavailability of energy and the starvation state. In: Kjelleberg S (ed) Starvation in bacteria. Plenum, New York, pp 1–23Google Scholar
  35. Munoz R, Diaz LF, Bordel S, Villaverde S (2007) Inhibitory effects of catechol accumulation on benzene biodegradation in Pseudomonas putida F1 cultures. Chemosphere 68:244–252. doi: 10.1016/j.chemosphere.2007.01.016 PubMedCrossRefGoogle Scholar
  36. Olsen RH, Kukor JJ, Kaphammer B (1994) A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1. J Bacteriol 176:3749–3756PubMedGoogle Scholar
  37. Olsen RH, Mikesell MD, Kukor JJ, Byrne AM (1995) Physiological attributes of microbial BTEX degradation in oxygen-limited environments. Environ Health Perspect 103(Suppl 5):49–51. doi: 10.2307/3432479 PubMedCrossRefGoogle Scholar
  38. Olsen RH, Kukor JJ, Byrne AM, Johnson GR (1997) Evidence for the evolution of a single component phenol/cresol hydroxylase from a multicomponent toluene monooxygenase. J Ind Microbiol Biotechnol 19:360–368. doi: 10.1038/sj.jim.2900453 PubMedCrossRefGoogle Scholar
  39. Park J, Chen YM, Kukor JJ, Abriola LM (2001) Influence of substrate exposure history on biodegradation in a porous medium. J Contam Hydrol 51:233–256. doi: 10.1016/S0169-7722(01)00125-5 PubMedCrossRefGoogle Scholar
  40. Park J, Malinverni J, Adriaens P, Kukor JJ (2003) Quantitative structure-activity relationship (QSAR) analysis of aromatic effector specificity in NtrC-like transcriptional activators from aromatic oxidizing bacteria. FEMS Microbiol Lett 224:45–52. doi: 10.1016/S0378-1097(03)00400-2 PubMedCrossRefGoogle Scholar
  41. Ramos JL, Duque E, Gallegos MT, Godoy P, Ramos-Gonzalez MI, Rojas A, Teran W, Segura A (2002) Mechanisms of solvent tolerance in Gram-negative bacteria. Annu Rev Microbiol 56:743–768. doi: 10.1146/annurev.micro.56.012302.161038 PubMedCrossRefGoogle Scholar
  42. Rüegg I, Hafner T, Bucheli-Witschel M, Egli T (2007) Dynamics of benzene and toluene degradation in Pseudomonas putida F1 in the presence of the alternative substrate succinate. Eng Life Sci 7:331–342. doi: 10.1002/elsc.200720202 CrossRefGoogle Scholar
  43. Shingler V (2003) Integrated regulation in response to aromatic compounds: from signal sensing to attractive behaviour. Environ Microbiol 5:1226–1241. doi: 10.1111/j.1462-2920.2003.00472.x PubMedCrossRefGoogle Scholar
  44. Sigg L, Stumm W (1996) Eine Einführung in die Chemie wässriger Lösungen und natürlicher Gewässer. Hochschulverlag an der ETH Zürich, ZürichGoogle Scholar
  45. Smith MR (1994) The physiology of aromatic hydrocarbon degrading bacteria. In: Ratledge C (ed) Biochemistry of microbial degradation. Kluwer, Dordrecht, pp 347–378Google Scholar
  46. Spormann AM, Widdel F (2000) Metabolism of alkylbenzenes, alkanes and other hydrocarbons in anaerobic bacteria. Biodegradation 11:85–105. doi: 10.1023/A:1011122631799 PubMedCrossRefGoogle Scholar
  47. Stumm W, Morgan JJ (1981) Aquatic chemistry: an introduction emphasizing chemical equilibria in natural waters. Wiley, New YorkGoogle Scholar
  48. Tao Y, Fishman A, Bentley WE, Wood TK (2004) Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1. Appl Environ Microbiol 70:3814–3820. doi: 10.1128/AEM.70.7.3814-3820.2004 PubMedCrossRefGoogle Scholar
  49. Tropel D, van der Meer JR (2004) Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500. doi: 10.1128/MMBR.68.3.474-500.2004 PubMedCrossRefGoogle Scholar
  50. Vroblesky DA, Chapelle FH (1994) Temporal and spatial changes of terminal electron accepting processes in a petroleum hydrocarbon-contaminated aquifer and the significance for contaminant biodegradation. Water Resour Res 30:1561–1570. doi: 10.1029/94WR00067 CrossRefGoogle Scholar
  51. Yerushalmi L, Manuel MF, Guiot SR (1999) Biodegradation of gasoline and BTEX in a microaerophilic biobarrier. Biodegradation 10:341–352. doi: 10.1023/A:1008327815105 PubMedCrossRefGoogle Scholar
  52. Yu H, Kim BJ, Rittmann BE (2001) The roles of intermediates in biodegradation of benzene, toluene and p-xylene by Pseudomonas putida F1. Biodegradation 12:455–463. doi: 10.1023/A:1015008627732 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Margarete Bucheli-Witschel
    • 1
  • Tina Hafner
    • 1
  • Irene Rüegg
    • 1
  • Thomas Egli
    • 1
  1. 1.Department of Environmental MicrobiologyEawag, Swiss Federal Institute of Aquatic Science and TechnologyDübendorfSwitzerland

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