, Volume 19, Issue 5, pp 749–757 | Cite as

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay

  • Kenzo Kubota
  • Daisuke Koma
  • Yoshiki Matsumiya
  • Seon-Yong Chung
  • Motoki Kubo
Original Paper


Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.


Cyclic alkane Car engine oil 2,6-DCPIP assay Hydrocarbon-degradation Long-chain hydrocarbon 


  1. Aislabie J, Saul DJ, Foght JM (2006) Bioremediation of hydrocarbon-contaminated polar soils. Extremophiles 10:171–179PubMedCrossRefGoogle Scholar
  2. Arenskötter M, Baumeister D, Berekka MM, Potter G, Kroppenstedt RM, Linos A, Steinbuchel A (2001) Taxonomic characterization of two rubber degrading bacteria belonging to the species Gordonia polyisoprenivorans and analysis of hyper variable regions of 16S rDNA sequenced. FEMS Microbiol Lett 205:277–282PubMedCrossRefGoogle Scholar
  3. Asperger O, Singh HD (1991) The biology of Acinetobacter. Plenum Press, New York, pp 323–350Google Scholar
  4. Dua M, Johri AK, Singh A, Sethunathan N (2002) Biotechnology and bioremediation: successes and limitations. Appl Microbiol Biotechnol 59:143–152PubMedCrossRefGoogle Scholar
  5. Hanson KG, Desai JD, Desai AJ (1993) A rapid and simple screening technique for potential crude oil degrading microorganisms. Biotechnol Tech 7:745–748CrossRefGoogle Scholar
  6. Iwamoto T, Nasu M (2001) Current bioremediation practice and perspective. J Biosci Bioeng 92:1–8PubMedCrossRefGoogle Scholar
  7. Ko SH, Lebeault JM (1999) Effect of a mixed culture on co-oxidation during the degradation of saturated hydrocarbon mixture. J Appl Microbiol 87:72–79PubMedCrossRefGoogle Scholar
  8. Koma D, Hasumi F, Yamamoto E, Ohta T, Chung SY, Kubo M (2001) Biodegradation of long-chain n-paraffines from waste oil of car engine by Acinetobacter sp. J Biosci Bioeng 91:94–96PubMedCrossRefGoogle Scholar
  9. Koma D, Hasumi F, Chung SY, Kubo M (2003a) Biodegradation of n-alkylcyclohexanes by co-oxidation via multiple pathways in Acinetobacter sp. ODDK71. J Biosci Bioeng 95:641–644PubMedGoogle Scholar
  10. Koma D, Sakashita Y, Kubota K, Fujii Y, Hasumi F, Chung SY, Kubo M (2003b) Degradation of car engine base oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A. Biosci Biochem Bioeng 67:1590–1593CrossRefGoogle Scholar
  11. Koma D, Sakashita Y, Kubota K, Fujii Y, Hasumi F, Chung SY, Kubo M (2005) Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48. Appl Microbiol Biotechnol 67:1590–1593Google Scholar
  12. Komukai-Nakamura S, Sugiura K, Yamauchi-Inomata Y, Toki H, Venkateswaran K, Yamamoto S, Tanaka H, Harayama S (1996) Construction of bacterial consortia that degrade Arabian light crude oil. J Ferment Bioeng 82:570–574CrossRefGoogle Scholar
  13. Konishi S, Ueda R (1992) Junkatsuyu no kiso to ouyou (in Japanese). Corona Publishing, Tokyo, pp 27–35Google Scholar
  14. Korda A, Santas P, Tenente A, Santas R (1997) Petroleum hydrocarbon bioremediation: sampling and analytical techniques, in situ treatments and commercial microorganisms currently used. Appl Microbiol Biotechnol 48:677–686PubMedCrossRefGoogle Scholar
  15. Kostichka K, Thomas SM, Gibson KJ, Nagarajan V, Cheng Q (2001) Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1. J Bacteriol 183:6478–6486PubMedCrossRefGoogle Scholar
  16. Kummer C, Schumann P, Stackebrandt E (1999) Gordonia alkanivorans sp. nov., isolated from tar-contaminated soil. Int J Syst Bacteriol 118:394–399Google Scholar
  17. Lal B, Khanna S (1996) Degradation of crude oil by Acinetobacter calcoaceticus and Alcaligenes odorans. J Appl Bacteriol 81:355–362PubMedGoogle Scholar
  18. Lappas AA, Patiaka D, Ikonomou D, Vasalos A (1997) Separation and characterization of paraffins and naphthenes from FCC feedstocks. Ind Eng Chem Res 36:3110–3115CrossRefGoogle Scholar
  19. Matsumiya Y, Kubo M (2007) Bioprocess handbook (in Japanese). NTS Publishing, Tokyo pp 638–652Google Scholar
  20. Perry JJ (1979) Microbial cooxidations involving hydrocarbons. Microbiol Rev 43:59–72PubMedGoogle Scholar
  21. Perry JJ (1984) Microbial metabolism of cyclic alkanes. Atlas, R. M., New York, pp 61–98Google Scholar
  22. Pritchard PH, Mueller IJG, 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
  23. Rehm H, Reiff I (1981) Mechanism and occurrence of microbial oxidation of long chain alkanes. Adv Biochem Eng 19:175–215Google Scholar
  24. Sakai Y, Maeng JH, Tani Y, Kato N (1994) Use of long-chain n-alkanes (C13-C44) by an isolate, Acinetobacter sp. M-1. Biosci Biotechnol Biochem 58:2128–2130CrossRefGoogle Scholar
  25. Sanpa S, Sumiyoshi S, Kujira T, Matsumiya Y, Kubo M (2006) Isolation and characterization of a Bluegill-degrading microorganism, and analysis of the root hair-promoting effect of the degraded products. Biosci Biotechnol Biochem 70:340–347PubMedCrossRefGoogle Scholar
  26. Schumacher JD, Fakoussa RM (1999) Degradation of alicyclic molecules by Rhodococcus ruber CD4. Appl Microbiol Biotechnol 52:85–90PubMedCrossRefGoogle Scholar
  27. Stroud JL, Paton GI, Semple KT (2007) Microbe-aliphatic hydrocarbon interactions in soil: implications for biodegradation and bioremediation. J Appl Microbiol 102:1239–1253PubMedCrossRefGoogle Scholar
  28. Throne-Holst M, Wentzel A, Ellingsen TE, Kotlar H-K, Zotchev SB (2007) Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874. Appl Environ Microbiol 73:3327–3332PubMedCrossRefGoogle Scholar
  29. van Beilen JB, Smits THM, Whyte LG, Schorcht S, Röthlisberger M, Plaggemeier T, Engesser KH, Witholt B (2002) Alkane hydroxylases homologues in Gram-positive strains. Environ Microbiol 4:676–682PubMedCrossRefGoogle Scholar
  30. van Beilen JB, Li Z, Duetz WA, Witholt B (2003) Diversity of alkane hydroxylase systems in the environment. Oil Gas Sci Technol 58:427–440CrossRefGoogle Scholar
  31. van Beilen JB, Funhoff EG (2007) Alkane hydroxylases involved in microbial alkane degradation. Appl Microbiol Biotechnol 74:13–21PubMedCrossRefGoogle Scholar
  32. van Hamme JD, Odumeru JA, Ward OP (2000) Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture. Can J Microbiol 46:441–450PubMedCrossRefGoogle Scholar
  33. Vidali M (2001) Bioremediation. A overview. Pure Appl Chem 73:1163–1172CrossRefGoogle Scholar
  34. Vomberg A, Klinner U (2000) Distribution of alkB genes within n-alkane-degrading bacteria. J Appl Microbiol 89:339–348PubMedCrossRefGoogle Scholar
  35. Whyte LG, Hawari J, Zhou E, Bourbonniére L, Inniss W, Greer CW (1998) Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp. Appl Environ Microbiol 64:2578–2584PubMedGoogle Scholar
  36. Whyte LG, Smits THM, Labbe D, Witholt B, Greer CW, van Beilen JB (2002) Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl Environ Microbiol 68:5933–5942PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Kenzo Kubota
    • 1
  • Daisuke Koma
    • 1
  • Yoshiki Matsumiya
    • 1
  • Seon-Yong Chung
    • 2
  • Motoki Kubo
    • 1
  1. 1.Department of Bioscience and Biotechnology, Faculty of Science and EngineeringRitsumeikan UniversityKusatsuJapan
  2. 2.Department of Environmental Engineering, College of EngineeringChonnam National UniversityKwangjuKorea

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