Skip to main content
SpringerLink
Log in

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

  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2

Similar content being viewed by others

References

  1. Altschul, SF, Gish, W, Miller, W, Myers, EW, Lipman, DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410

    Article  PubMed  CAS  Google Scholar 

  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: 3977–3984

    Article  PubMed  CAS  Google Scholar 

  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: 1439–1448

    Article  CAS  Google Scholar 

  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: 3376–3380

    Article  PubMed  CAS  Google Scholar 

  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: 3–15

    Article  PubMed  CAS  Google Scholar 

  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: 6597–6604

    Article  PubMed  CAS  Google Scholar 

  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: 197–212

    Article  Google Scholar 

  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: 47–60

    Article  Google Scholar 

  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: 760–768

    Article  PubMed  CAS  Google Scholar 

  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: 191–197

    Article  PubMed  CAS  Google Scholar 

  11. Heid, CA, Stevens, J, Livak, KJ, Williams, PM (1996) Real time quantitative PCR. Genome Res 6: 986–994

    PubMed  CAS  Google Scholar 

  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: 1392–1403

    Article  PubMed  CAS  Google Scholar 

  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: 327–335

    Article  PubMed  CAS  Google Scholar 

  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: 5154–5160

    Article  PubMed  CAS  Google Scholar 

  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: 323–333

    Article  PubMed  CAS  Google Scholar 

  16. MacKay, IM (2004) Real-time PCR in the microbiology laboratory. Clin Microb Infect 10: 190–212

    Article  CAS  Google Scholar 

  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: 1008–1016

    Article  PubMed  CAS  Google Scholar 

  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: 2011–2017

    Article  PubMed  CAS  Google Scholar 

  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: 5537–5548

    Article  PubMed  CAS  Google Scholar 

  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: 517–522

    Article  PubMed  CAS  Google Scholar 

  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: 369–378

    Article  PubMed  CAS  Google Scholar 

  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: 804–815

    Article  PubMed  CAS  Google Scholar 

  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: 89–98

    Article  PubMed  CAS  Google Scholar 

  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: 155–164

    Article  PubMed  CAS  Google Scholar 

  25. Van Beilen, JB, Wubbolts, MG, Witholt, B (1994) Genetics of alkane oxidation by Pseudomonas olevorans. Biodegradation 5: 161–174

    Article  PubMed  Google Scholar 

  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: 1621–1630

    PubMed  Google Scholar 

  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: 174–182

    Article  PubMed  Google Scholar 

  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: 59–65

    Article  PubMed  CAS  Google Scholar 

  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: 5933–5942

    Article  PubMed  CAS  Google Scholar 

  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: 141–150

    CAS  PubMed  Google Scholar 

  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: 252–258

    Article  PubMed  CAS  Google Scholar 

  32. Zhang, T, Fang, HHP (2006) Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Appl Microbiol Biotechnol 70: 281–289

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Tasmanian Institute of Agricultural Research, University of Tasmania, Hobart, 7001, Australia

    Shane M. Powell & John P. Bowman

  2. Department of Heritage and Environment, Australian Antarctic Division, 203 Channel Highway, Kingston, 7050, Australia

    Shane M. Powell, Susan H. Ferguson & Ian Snape

Authors
  1. Shane M. Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susan H. Ferguson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John P. Bowman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ian Snape
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shane M. Powell.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Powell, S.M., Ferguson, S.H., Bowman, J.P. et al. Using Real-Time PCR to Assess Changes in the Hydrocarbon-Degrading Microbial Community in Antarctic Soil During Bioremediation. Microb Ecol 52, 523–532 (2006). https://doi.org/10.1007/s00248-006-9131-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-006-9131-z

Keywords

Search

Navigation