Applied Microbiology and Biotechnology

, Volume 67, Issue 2, pp 275–285 | Cite as

Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1

  • Yong-Hak Kim
  • James P. Freeman
  • Joanna D. Moody
  • Karl-Heinrich Engesser
  • Carl E. Cerniglia
Environmental Biotechnology


The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (μ′=0.058 h−1) than at pH 6.5 (μ′=0.028 h−1). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2′-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.



This research was supported in part by an appointment to the Postgraduate Research Participation Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the United States Department of Energy and the Food and Drug Administration


  1. Ahn Y, Sanseverino J, Sayler GS (1999) Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils. Biodegradation 10:149–157CrossRefPubMedGoogle Scholar
  2. Atlas RM (1991) Microbial hydrocarbon degradation–bioremediation of oil spills. J Chem Technol Biotechnol 52:149–156Google Scholar
  3. Boldrin B, Tiehm A, Fritsche C (1993) Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by Mycobacterium sp. Appl Environ Microbiol 59:1927–1930PubMedGoogle Scholar
  4. Bolton JL, Michael MA, Penning TM, Dryhurst G, Monks TJ (2000) Role of quinones in toxicity. Chem Res Toxicol 13:135–160PubMedGoogle Scholar
  5. Brennan PJ, Nikaido H (1995) The envelope of mycobacteria. Annu Rev Biochem 64:29–63CrossRefPubMedGoogle Scholar
  6. Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3:351–368Google Scholar
  7. Chevalier J, Pommier MT, Cremieux A, Michel G (1998) Influence of Tween 80 on the mycolic acid composition of three cutaneous corynebacteria. J Gen Microbiol 134:2457–2461Google Scholar
  8. Churchill SA, Harper JP, Churchill PF (1999) Isolation and characterization of a Mycobacterium species capable of degrading three- and four-ring aromatic and aliphatic hydrocarbons. Appl Environ Microbiol 65:549–552PubMedGoogle Scholar
  9. Dean-Ross D, Cerniglia CE (1996) Degradation of pyrene by Mycobacterium flavescens. Appl Microbiol Biotechnol 46:307–312CrossRefPubMedGoogle Scholar
  10. Fritzsche C (1994) Degradation of pyrene at low defined oxygen concentrations by a Mycobacterium sp. Appl Environ Microbiol 60:1687–1689PubMedGoogle Scholar
  11. Grosser RJ, Warshawsky D, Vestal JB (1991) Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils. Appl Environ Microbiol 57:3462–3469PubMedGoogle Scholar
  12. Harvey RG (1996) Mechanisms of carcinogenesis of polycyclic aromatic hydrocarbons. Polycyclic Aromat Compounds 9:1–23Google Scholar
  13. Heitkamp MA, Franklin W, Cerniglia CE (1988a) Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium. Appl Environ Microbiol 54:2549–2555PubMedGoogle Scholar
  14. Heitkamp MA, Freeman JP, Miller DW, Cerniglia CE (1988b) Pyrene degradation by a Mycobacterium sp.: identification of ring oxidation and ring fission products. Appl Environ Microbiol 54:2556–2565PubMedGoogle Scholar
  15. Heitkamp MA, Cerniglia CE (1989) Polycyclic aromatic hydrocarbon degradation by a Mycobacterium sp. in microcosms containing sediment and water from a pristine ecosystem. Appl Environ Microbiol 55:1968–1973PubMedGoogle Scholar
  16. Jackson M, Raynaud C, Laneelle MA, Guilhot C, Laurent-Winter C, Ensergueix D, Gicquel B, Daffe M (1999) Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol Microbiol 31:1573–1587CrossRefPubMedGoogle Scholar
  17. Keith LH, Telliard WA (1979) Priority pollutants I A perspective view. Environ Sci Technol 13:416–423Google Scholar
  18. Kelley I, Freeman JP, Evans FE, Cerniglia CE (1993) Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1. Appl Environ Microbiol 59:800–806PubMedGoogle Scholar
  19. Kim YH, Engesser KH, Cerniglia CE (2003) Two polycyclic aromatic hydrocarbon o-quinone reductases from a pyrene-degrading Mycobacterium. Arch Biochem Biophys 416:209–217PubMedGoogle Scholar
  20. Kim YH, Moody JD, Freeman JP, Brezna B, Engesser KH, Cerniglia CE (2004) Evidence for the existence of PAH-quinone reductase and catechol-O-methyltransferase in Mycobacterium vanbaalenii PYR-1. J Ind Microbiol Biotechnol (in press)Google Scholar
  21. Korycka-Machala M, Ziolkowski A, Rumijowska-Galewicz A, Lisowska K, Sedlaczek L (2001) Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds. Microbiology 147:2769–2781PubMedGoogle Scholar
  22. Kremer L, Guerardel Y, Gurcha SS, Locht C, Besra GS (2002) Temperature-induced changes in the cell-wall components of Mycobacterium thermoresistibile. Microbiology 148:3145–3154PubMedGoogle Scholar
  23. Molina M, Araujo R, Hodson RR (1999) Cross-induction of pyrene and phenanthrene in a Mycobacterium sp. isolated from polycyclic aromatic hydrocarbon contaminated river sediments. Can J Microbiol 45:520–529CrossRefPubMedGoogle Scholar
  24. Moody JD, Freeman JP, Doerge DR, Cerniglia CE (2001) Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1. Appl Environ Microbiol 67:1476–1483CrossRefPubMedGoogle Scholar
  25. Moody JD, Fu PP, Freeman JP, Cerniglia CE (2003) Regio- and stereoselective metabolism of 7,12-dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1. Appl Environ Microbiol 69:3924–3931CrossRefPubMedGoogle Scholar
  26. Moody JD, Freeman JP, Fu PP, Cerniglia CE (2004) Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1. Appl Environ Microbiol 70:340–345CrossRefPubMedGoogle Scholar
  27. Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, Zeiger E (1986) Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ Mutagen 8 [Suppl 7]:1–119Google Scholar
  28. Pothuluri JV, Cerniglia CE (1994) Microbial metabolism of polycyclic aromatic hydrocarbons. In: Chaudhry GR (ed) Biological degradation and bioremediation of toxic chemicals. Kluwer, Dordrecht, pp 92–124Google Scholar
  29. Rehmann K, Noll HP, Steinberg CEW, Kettrup AA (1998) Pyrene degradation by Mycobacterium sp. strain KR2. Chemosphere 36:2977–2992CrossRefPubMedGoogle Scholar
  30. Schneider J, Grosser R, Jayasimhulu K, Xue W, Warshawsky D (1996) Degradation of pyrene, benz[a]anthacene, benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site. Appl Environ Microbiol 62:13–19PubMedGoogle Scholar
  31. Stingley RL, Khan AA, Cerniglia CE (2004) Molecular characterization of a phenanthrene degradation pathway in Mycobacterium vanbaalenii PYR-1. Biochem Biophys Res Commun 322:133–146CrossRefPubMedGoogle Scholar
  32. Stratton HM, Brooks PR, Carr EL, Seviour RJ (2003) Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp from activated sludgefoams. Syst Appl Microbiol 26:165–171CrossRefPubMedGoogle Scholar
  33. Sutherland JB, Rafii F, Khan AA, Cerniglia CE (1995) Mechanisms of polycyclic aromatic hydrocarbon degradation. In: Young LY, Cerniglia CE (eds) Microbial transformation and degradation of toxic organic chemicals. Wiley–Liss, New York, pp 269–306Google Scholar
  34. Tiehm A (1994) Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants. Appl Environ Microbiol 60:258–263PubMedGoogle Scholar
  35. Vila J, López Z, Sabaté J, Minguillón C, Solanas AM, Grifoll M (2001) Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons. Appl Environ Microbiol 67:5497–5505CrossRefPubMedGoogle Scholar
  36. Wick LY, Wattiau P, Harms H (2002) Influence of the growth substrate on the mycolic acid profiles of mycobacteria. Environ Microbiol 4:612–616CrossRefPubMedGoogle Scholar
  37. Zylstra GJ, Wang XP, Kim E, Didolkar VA (1994) Cloning and analysis of the genes for polycyclic aromatic hydrocarbon degradation. Ann NY Acad Sci 721:386–398PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Yong-Hak Kim
    • 1
  • James P. Freeman
    • 2
  • Joanna D. Moody
    • 1
  • Karl-Heinrich Engesser
    • 3
  • Carl E. Cerniglia
    • 1
  1. 1.Division of Microbiology, National Center for Toxicological ResearchUnited States Food and Drug AdministrationJeffersonUSA
  2. 2.Division of Chemistry, National Center for Toxicological ResearchUnited States Food and Drug AdministrationJeffersonUSA
  3. 3.Abteilung Biologische Abluftreinigung, ISWAUniversität StuttgartStuttgartGermany

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