Skip to main content
SpringerLink
Log in

Effect of Incubation Conditions on the Enrichment of Pyrene-degrading Bacteria Identified by Stable-isotope Probing in an Aged, PAH-contaminated Soil

  • Original Article
  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

To determine whether the diversity of pyrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon-contaminated soil is affected by the addition of inorganic nutrients or by slurrying the soil, various incubation conditions (all including phosphate buffer) were examined by mineralization studies and stable-isotope probing (SIP). The addition of nitrogen to either continuously mixed slurry or static field-wet soil incubations increased the rate and extent of mineralization of [14C]pyrene, with the most rapid mineralization observed in slurried, nitrogen-amended soil. Microcosms of slurry and static field-wet soil amended with nitrogen were also examined by SIP with [U-13C]pyrene. Recovered 13C-enriched deoxyribonucleic acid (DNA) was analyzed by denaturing-gradient gel electrophoresis (DGGE) and 16S ribosomal ribonucleic acid (rRNA) gene clone libraries. DGGE profiles of 13C-enriched DNA fractions from both incubation conditions were similar, suggesting that pyrene-degrading bacterial community diversity may be independent of treatment method. The vast majority (67 of 71) of the partial sequences recovered from clone libraries were greater than or equal to 97% similar to one another, 98% similar to sequences of pyrene-degrading bacteria previously detected by SIP with pyrene in different soil, and only 89% similar to the closest cultivated genus. All of the sequences recovered from the field-wet incubation and most of the sequences recovered from the slurry incubation were in this clade. Of the four sequences from slurry incubations not within this clade, three possessed greater than 99% similarity to the 16S rRNA gene sequences of phylogenetically dissimilar Caulobacter spp.

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
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Abraham W, Strompl C, Meyer H, Lindholst S, Moore ERB, Christ R, Vancanneyt M, Tindall BJ, Bennasar A, Smit J, Tesar M (1999) Phylogeny and polyphasic taxonomy of Caulobacter species. Proposal of Maricaulis gen. nov. with Maricaulis Maris (Poindexter) comb. nov. as the type species, and emended description of the genera Brevundimonas and Caulobacter. Intl J Syst Bacteriol 49:1053–1073

    CAS  Google Scholar 

  2. Agency for Toxic Substances and Disease Registry (1995) Public health statement for polycyclic aromatic hydrocarbons. US Department of Health and Human Services, Public Health Service, Atlanta, GA

    Google Scholar 

  3. Aitken MD, Stringfellow WT, Nagel RD, Kazunga C, Chen SH (1998) Characteristics of phenanthrene-degrading bacteria isolated from soils contaminated with polycyclic aromatic hydrocarbons. Can J Microbiol 44:743–752

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  5. Borneman J, Hartin RJ (2000) PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360

    Article  PubMed  CAS  Google Scholar 

  6. Bouyoucos GJ (1962) Hydrometer method improved for making particle size analysis of soils. Agron J 54:464–465

    Article  Google Scholar 

  7. Carmichael LM, Pfaender FK (1997) The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils. Biodegradation 8:1–13

    Article  PubMed  CAS  Google Scholar 

  8. Cassel DK, Nielsen DR (1986) Field capacity and available water capacity. In: Klute A (ed) Methods of soil analysis. Part 1. Physical and mineralogical methods. Agronomy monograph no. 9. 2nd edn. American Society of Agronomy, Wisconsin, pp 901–926

    Google Scholar 

  9. Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3:351–368

    Article  CAS  Google Scholar 

  10. Cheung P, Kinkle BK (2001) Mycobacterium diversity and pyrene mineralization in petroleum-contaminated soils. Appl Environ Microbiol 67:2222–2229

    Article  PubMed  CAS  Google Scholar 

  11. Cole JR, Chai B, Marsh TL, Farris RJ, Wang Q, Kulam SA, Chandra S, McGarrell DM, Schmidt TM, Garrity GM, Tiedje JM (2003) The ribosomal database project (RDP-II): Previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31:442–443

    Article  PubMed  CAS  Google Scholar 

  12. Dean-Ross D, Cerniglia CE (1996) Degradation of pyrene by Mycobacterium flavescens. Appl Microbiol Biotechnol 46:307–312

    Article  PubMed  CAS  Google Scholar 

  13. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853

    Article  PubMed  CAS  Google Scholar 

  14. El Fantroussi S, Verschuere L, Verstraete W, Top EM (1999) Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16 S rRNA gene fingerprints and community-level physiological profiles. Appl Environ Microbiol 65:982–988

    PubMed  Google Scholar 

  15. Eriksson M, Dalhammar G, Mohn WW (2002) Bacterial growth and biofilm production on pyrene. FEMS Microbiol Ecol 40:21–27

    Article  CAS  PubMed  Google Scholar 

  16. Guthrie EA, Pfaender FK (1998) Reduced pyrene bioavailability in microbially active soils. Environ Sci Technol 32:501–508

    Article  CAS  Google Scholar 

  17. Heitkamp MA, Franklin W, Cerniglia CE (1998) Microbial metabolism of polycyclic aromatic hydrocarbons: Isolation and characterization of a pyrene-degrading bacterium. Appl Environ Microbiol 54:2549–2555

    Google Scholar 

  18. Huesemann MH, Hausmann TS, Fortman TJ (2004) Does bioavailability limit biodegradation? A comparison of hydrocarbon biodegradation and desorption rates in aged soils. Biodeg 15:261–274

    Article  PubMed  CAS  Google Scholar 

  19. Hwang S, Cutright TJ (2002) Biodegradability of aged pyrene and phenanthrene in a natural soil. Chemosphere 47:891–899

    Article  PubMed  CAS  Google Scholar 

  20. Hwang S, Cutright TJ (2003) Preliminary exploration of the relationships between soil characteristics and PAH desorption and biodegradation. Environ Intl 29:887–894

    Article  CAS  Google Scholar 

  21. Jeon CO, Park W, Padmanabhan P, DeRito C, Snape JR, Madsen EL (2003) Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc Natl Acad Sci USA 100:13591–13596

    Article  PubMed  CAS  Google Scholar 

  22. Kanaly RA, Harayama S (2000) Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by bacteria. J Bacteriol 182:2059–2067

    Article  PubMed  CAS  Google Scholar 

  23. Kazunga C, Aitken MD (2000) Products from the incomplete metabolism of pyrene by polycyclic aromatic hydrocarbon-degrading bacteria. Appl Environ Microbiol 66:1917–1922

    Article  PubMed  CAS  Google Scholar 

  24. Kim Y, Freeman JP, Moody JD, Engesser K, Cerniglia CE (2005) Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1. Appl Microbiol Biotechnol 67:275–285

    Article  PubMed  CAS  Google Scholar 

  25. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid sequencing techniques in bacterial systematics. Wiley, New York, pp 115–175

    Google Scholar 

  26. MacLeod CT, Daugulis AJ (2003) Biodegradation of polycyclic aromatic hydrocarbons in a two-phase partitioning bioreactor in the presence of a bioavailable solvent. Appl Microbiol Biotechnol 62:291–296

    Article  PubMed  CAS  Google Scholar 

  27. Miller CD, Hall K, Liang YN, Nieman K, Sorensen D, Issa B, Anderson AJ, Sims RC (2004) Isolation and characterization of polycyclic aromatic hydrocarbon-degrading Mycobacterium isolates from soil. Microb Ecol 48:230–238

    Article  PubMed  CAS  Google Scholar 

  28. Mueller JG, Devereux R, Santavy DL, Lantz SE, Willis SG, Pritchard PH (1997) Phylogenetic and physiological comparisons of PAH-degrading bacteria from geographically diverse soils. Antonie van Leeuwenhoek 71:329–343

    Article  PubMed  CAS  Google Scholar 

  29. Mueller JG, Lank SE, Blattmann BO, Chapman PJ (1991) Bench-scale evaluation of alternative biological treatment processes for the remediation of pentachlorophenol- and creosote-contaminated materials: Slurry-phase bioremediation. Environ Sci Technol 25:1055–1061

    Article  CAS  Google Scholar 

  30. Northcott GL, Jones KC (2000) Developing a standard spiking procedure for the introduction of hydrophobic organic compounds into field-wet soil. Environ Toxicol Chem 19:2409–2417

    Article  CAS  Google Scholar 

  31. Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Computer Appl Biosci 12:357–358

    CAS  Google Scholar 

  32. Parrish ZD, White JC, Isleyen M, Gent MPN, Iannucci-Berger W, Eitzer BD, Kelsey JW, Mattina MI (2006) Accumulation of weathered polycyclic aromatic hydrocarbons (PAHs) by plant and earthworm species. Chemosphere 64:609–618

    Article  PubMed  CAS  Google Scholar 

  33. Poindexter JS (1981) The Caulobacters: ubiquitous unusual bacteria. Microbiol Rev 45:123–179

    PubMed  CAS  Google Scholar 

  34. Potter CL, Glaser JA, Chang LW, Meier JR, Dosani MA, Herrmann RF (1999) Degradation of polynuclear aromatic hydrocarbons under bench-scale compost conditions. Environ Sci Technol 33:1717–1725

    Article  CAS  Google Scholar 

  35. Ramirez N, Cutright T, Ju L (2001) Pyrene biodegradation in aqueous solutions and soil slurries by Mycobacterium PYR-1 and enriched consortium. Chemosphere 44:1079–1086

    Article  PubMed  CAS  Google Scholar 

  36. Rappe MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394

    Article  PubMed  CAS  Google Scholar 

  37. Raskin L, Stromley JM, Rittman BE, Stahl DA (1994) Group-specific 16 S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 60:1232–1240

    PubMed  CAS  Google Scholar 

  38. Rehmann K, Noll HP, Steinberg CEW, Kettrup AA (1998) Pyrene degradation by Mycobacterium sp. strain KR2. Chemosphere 36:2977–2992

    Article  PubMed  CAS  Google Scholar 

  39. Reid BJ, Northcott GL, Jones KC, Semple KT (1998) Evaluation of spiking procedures for the introduction of poorly water soluble contaminants into soil. Environ Sci Technol 32:3224–3227

    Article  CAS  Google Scholar 

  40. Risk Reduction Engineering Laboratory, US EPA (1993) Pilot-scale demonstration of a slurry-phase biological reactor for creosote-contaminated soil: applications analysis report. EPA/540/A5-91/009

  41. 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 

  42. Sabat G, Rose P, Hickey WJ, Harkin JM (2000) Selective and sensitive method for PCR amplification of Escherichia coli 16 S rRNA genes in soil. Appl Environ Microbiol 66:844–849

    Article  PubMed  CAS  Google Scholar 

  43. Schulte EE, Hopkins BG (1996) Estimation of soil organic matter by weight loss-on-ignition. In: Magdoff FR, Tabatabai MA, Hanlon EA Jr (eds) Soil organic matter: analysis and interpretation. Soil Science Society of America, Wisconsin, pp 21–31

    Google Scholar 

  44. Singleton DR, Powell SN, Sangaiah R, Gold A, Ball LM, Aitken MD (2005) Stable-isotope probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating contaminated soil. Appl Environ Microbiol 71:1202–1209

    Article  PubMed  CAS  Google Scholar 

  45. Singleton DR, Sangaiah R, Gold A, Ball LM, Aitken MD (2006) Identification and quantification of uncultivated proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil. Environ Microbiol 8:1736–1745

    Article  PubMed  CAS  Google Scholar 

  46. Sly LI, Cox TL, Beckenham TB (1999) The phylogenetic relationships of Caulobacter, Asticcacaulis and Brevundimonas species and their taxonomic implications. Intl J Syst Bacteriol 49:483–488

    Article  Google Scholar 

  47. Takai K, Horikoshi K (2000) Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes. Appl Environ Microbiol 66:5066–5072

    Article  PubMed  CAS  Google Scholar 

  48. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  49. US EPA Office of Solid Waste and Emergency Response (1999) Use of monitored natural attenuation at Superfund, RCRA corrective action, and underground storage tank sites. OSWER Directive 9200.4-17P

    Google Scholar 

  50. Viñas M, Sabaté J, Espuny MJ, Solanas AM (2005) Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil. Appl Environ Microbiol 71:7008–7018

    Article  PubMed  CAS  Google Scholar 

  51. Walter U, Beyer M, Klein J, Rehm H (1991) Degradation of pyrene by Rhodococcus sp. UW1. Appl Microbiol Biotechnol 34:671–676

    Article  CAS  Google Scholar 

  52. Watson ME, Brown JR (1998) pH and lime requirement. In: Brown JR (ed) Recommended chemical test procedures for north central region, NCR Publication no. 221 (revised), Missouri Agr 0045p Sta SB 1001. NCR, Missouri, pp 13–16

    Google Scholar 

  53. White JC, Alexander M, Pignatello JJ (1999) Enhancing the bioavailability of organic compounds sequestered in soil and aquifer solids. Environ Toxicol Chem 18:182–187

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the National Institute of Environmental Health Sciences Superfund Basic Research Program (5 P42 ES05948) and by the National Science Foundation (BES 0221836). MDJ was supported by the Research Education Support Program, a project of the National Science Foundation Alliance for Graduate Education and the Professoriate (HRD0450099).

Author information

Authors and Affiliations

  1. Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, CB no. 7431, Chapel Hill, NC, 27599-7431, USA

    Maiysha D. Jones, David R. Singleton, Darryl P. Carstensen, Sabrina N. Powell, Julie S. Swanson, Frederic K. Pfaender & Michael D. Aitken

Authors
  1. Maiysha D. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David R. Singleton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Darryl P. Carstensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sabrina N. Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Julie S. Swanson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frederic K. Pfaender
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael D. Aitken
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael D. Aitken.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jones, M.D., Singleton, D.R., Carstensen, D.P. et al. Effect of Incubation Conditions on the Enrichment of Pyrene-degrading Bacteria Identified by Stable-isotope Probing in an Aged, PAH-contaminated Soil. Microb Ecol 56, 341–349 (2008). https://doi.org/10.1007/s00248-007-9352-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-007-9352-9

Keywords

Search

Navigation