Microbial Ecology

, Volume 52, Issue 3, pp 523–532 | Cite as

Using Real-Time PCR to Assess Changes in the Hydrocarbon-Degrading Microbial Community in Antarctic Soil During Bioremediation

  • Shane M. PowellEmail author
  • Susan H. Ferguson
  • John P. Bowman
  • Ian Snape


A real-time polymerase chain reaction (PCR) method to quantify the proportion of microorganisms containing alkane monooxygenase was developed and used to follow changes in the microbial community in hydrocarbon-contaminated Antarctic soil during a bioremediation field trial. Assays for the alkB and rpoB genes were validated and found to be both sensitive and reproducible (less than 2% intrarun variation and 25–38% interrun variation). Results from the real-time PCR analysis were compared to analysis of the microbial population by a culture-based technique [most probable number (MPN) counts]. Both types of analysis indicated that fertilizer addition to hydrocarbon-contaminated soil stimulated the indigenous bacterial population within 1 year. The proportion of alkB containing microorganisms was positively correlated to the concentration of n-alkanes in the soil. After the concentration of n-alkanes in the soil decreased, the proportion of alkane-degrading microorganisms decreased, but the proportion of total hydrocarbon-degrading microorganisms increased, indicating another shift in the microbial community structure and ongoing biodegradation.


Much Probable Number rpoB Gene Antarctic Soil alkB Gene Competitive Polymerase Chain Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to thank Paul McA. Harvey (Australian Antarctic Division) for carrying out the chemical analysis of the soil samples. This work was supported by AAS grant 1163.


  1. 1.
    Altschul, SF, Gish, W, Miller, W, Myers, EW, Lipman, DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410PubMedCrossRefGoogle Scholar
  2. 2.
    Beller, HR, Kane, SR, Legler, TC, Alvarez, PJJ (2002) A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon degrading bacteria based on a catabolic gene. Environ Sci Technol 36: 3977–3984PubMedCrossRefGoogle Scholar
  3. 3.
    Coulon, F, Pelletier, E, Gourhant, L, Delille, D (2005) Effects of nutrient and temperature on degradation of petroleum hydrocarbons in contaminated sub-Antarctic soil. Chemosphere 10: 1439–1448CrossRefGoogle Scholar
  4. 4.
    Dahllof, I, Baillie, H, Kjelleberg, S (2000) rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl Environ Microbiol 66: 3376–3380PubMedCrossRefGoogle Scholar
  5. 5.
    Devers, M, Soulas, G, Martin-Laurent, F (2004) Real-time reverse transcription PCR analysis of expression of atrazine catabolism genes in two bacterial strains isolated from soil. J Microbiol Methods 56: 3–15PubMedCrossRefGoogle Scholar
  6. 6.
    Dionisi, HM, Harms, G, Layton, AC, Gregory, IR, Parker, J, Hawkins, SA, Robinson, KG, Sayler, GS (2003) Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction. Appl Environ Microbiol 69: 6597–6604PubMedCrossRefGoogle Scholar
  7. 7.
    Ferguson, SH, Franzmann, PD, Revill, AT, Snape, I, Rayner, JL (2003) The effects of nitrogen and water on mineralisation of hydrocarbons in diesel-contaminated terrestrial Antarctic. Cold Reg Sci Technol 37: 197–212CrossRefGoogle Scholar
  8. 8.
    Ferguson, SH, Woinarski, AZ, Snape, I, Morris, CE, Revill, AT (2004) A field trial of in situ chemical oxidation to remediate long-term diesel contaminated Antarctic soil. Cold Reg Sci Technol 40: 47–60CrossRefGoogle Scholar
  9. 9.
    Gruntzig, V, Nold, SC, Zhou, J, Tiedje, JM (2001) Pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time PCR. Appl Environ Microbiol 67: 760–768PubMedCrossRefGoogle Scholar
  10. 10.
    Hara, A, Baik, S, Syutsubo, K, Misawa, N, Smits, THM, van Beilen, JB, Harayama, S (2004) Cloning and functional analysis for alkB genes in Alcanivorax borkumensis SK2. Environ Microbiol 6: 191–197PubMedCrossRefGoogle Scholar
  11. 11.
    Heid, CA, Stevens, J, Livak, KJ, Williams, PM (1996) Real time quantitative PCR. Genome Res 6: 986–994PubMedGoogle Scholar
  12. 12.
    Heiss-Blanquet, S, Benoit, Y, Marechaux, C, Monot, F (2005) Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR. J Appl Microbiol 99: 1392–1403PubMedCrossRefGoogle Scholar
  13. 13.
    Henry, S, Baudoin, E, López-Gutiérrez, JC, Martin-Laurent, F, Brauman, A, Philippot, L (2004) Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J Microbiol Methods 59: 327–335PubMedCrossRefGoogle Scholar
  14. 14.
    Hristova, KR, Lutenegger, CM, Scow, KM (2001) Detection and quantification of methyl tert-butyl ether degrading strain PM1 by real time TaqMan PCR. Appl Environ Microbiol 67: 5154–5160PubMedCrossRefGoogle Scholar
  15. 15.
    Luz, AP, Pellzari, VH, Whyte, LG, Greer, CW (2004) A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil. Can J Microbiol 50: 323–333PubMedCrossRefGoogle Scholar
  16. 16.
    MacKay, IM (2004) Real-time PCR in the microbiology laboratory. Clin Microb Infect 10: 190–212CrossRefGoogle Scholar
  17. 17.
    Okano, Y, Hristova, KR, Leutenegger, CM, Jackson, LE, Denison, RF, Gebreyesus, B, Lebauer, D, Scow, KM (2004) Application of real-time PCR to study effects of ammonium on population size of ammonia-oxidizing bacteria in soil. Appl Environ Microbiol 70: 1008–1016PubMedCrossRefGoogle Scholar
  18. 18.
    Powell, SM, Ferguson, SH, Snape, I, Siciliano, SD (2006) Fertilisation stimulates anaerobic fuel degradation of Antarctic soils by denitrifying organisms. Environ Sci Technol 40: 2011–2017PubMedCrossRefGoogle Scholar
  19. 19.
    Roling, WFM, Milner, MG, Jones, DM, Lee, K, Daniel, F, Swannell, RJP, Head, IM (2002) Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Appl Environ Microbiol 68: 5537–5548PubMedCrossRefGoogle Scholar
  20. 20.
    Sei, K, Sugimoto, Y, Mori, K, Maki, H, Kohno, T (2003) Monitoring of alkane-degrading bacteria in a sea-water microcosm during crude oil degradation by polymerase chain reaction based on alkane-catabolic genes. Environ Microbiol 5: 517–522PubMedCrossRefGoogle Scholar
  21. 21.
    Smits, THM, Devenoges, C, Szynalski, K, Maillard, J, Holliger, C (2004) Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides and Desulfitobacterium in microbial communities. J Microbiol Methods 57: 369–378PubMedCrossRefGoogle Scholar
  22. 22.
    Smith, CJ, Nedwell, DB, Dong, LF, Osborn, AM (2006) Evaluation of quantitative polymerase chain reaction based approaches for determining gene copy and gene transcript numbers in environmental samples. Environ Microbiol 8: 804–815PubMedCrossRefGoogle Scholar
  23. 23.
    Snape, I, Ferguson, SH, Harvey, PMcA, Riddle, MJ (2006) Investigation of evaporation and biodegradation of fuel spills in Antarctica: II—extent of natural attenuation at Casey Station. Chemosphere 63: 89–98PubMedCrossRefGoogle Scholar
  24. 24.
    Stubner, S (2002) Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection. J Microbiol Methods 50: 155–164PubMedCrossRefGoogle Scholar
  25. 25.
    Van Beilen, JB, Wubbolts, MG, Witholt, B (1994) Genetics of alkane oxidation by Pseudomonas olevorans. Biodegradation 5: 161–174PubMedCrossRefGoogle Scholar
  26. 26.
    Van Beilen, JB, Panke, S, Lucchini, S, Franchini, AG, Rothlisberger, M, Withholt, B (2001) Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology 147: 1621–1630PubMedGoogle Scholar
  27. 27.
    Van Beilen, JB, Mourlane, F, Seeger, MA, Kovac, J, Li, Z, Smits, TH, Fritsche, U, Witholt, B (2003) Cloning of Baeyer–Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ Microbiol 5: 174–182PubMedCrossRefGoogle Scholar
  28. 28.
    Van Beilen, JB, Funhoff, EG, van Loon, A, Just, A, Kaysser, L, Bouza, M, Holtackers, R, Rothlisberger, M, Li, Z, Witholt, B (2006) Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases. Appl Environ Microbiol 72: 59–65PubMedCrossRefGoogle Scholar
  29. 29.
    Whyte, LG, Smits, THM, Labbe, D, Witholt, B, Greer, CW, Van Beilen, JB (2002a) Gene cloning and characterisation of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl Environ Microbiol 68: 5933–5942PubMedCrossRefGoogle Scholar
  30. 30.
    Whyte, LG, Schultz, A, Van Beilen, JB, Luz, AP, Pellizari, V, Labbe, D, Greer, CW (2002b) Prevalence of alkane mono-oxygenase genes in Arctic and Antarctic hydrocarbon-contaminated and pristine soils. FEMS Microbiol Ecol 41: 141–150PubMedGoogle Scholar
  31. 31.
    Wrenn, BA, Venosa, AD (1996) Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. Can J Microbiol 42: 252–258PubMedCrossRefGoogle Scholar
  32. 32.
    Zhang, T, Fang, HHP (2006) Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Appl Microbiol Biotechnol 70: 281–289PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Shane M. Powell
    • 1
    • 2
    Email author
  • Susan H. Ferguson
    • 2
  • John P. Bowman
    • 1
  • Ian Snape
    • 2
  1. 1.Tasmanian Institute of Agricultural ResearchUniversity of TasmaniaHobartAustralia
  2. 2.Department of Heritage and EnvironmentAustralian Antarctic DivisionKingstonAustralia

Personalised recommendations