Register      Login
Soil Research Soil Research Society
Soil, land care and environmental research
RESEARCH ARTICLE

Microbial community and ecotoxicity analysis of bioremediated, weathered hydrocarbon-contaminated soil

Petra J. Sheppard A B , Eric M. Adetutu A , Tanvi H. Makadia A and Andrew S. Ball A
+ Author Affiliations
- Author Affiliations

A School of Biological Sciences, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia.

B Corresponding author. Email: shep0131@flinders.edu.au

Soil Research 49(3) 261-269 https://doi.org/10.1071/SR10159
Submitted: 2 August 2010  Accepted: 11 November 2010   Published: 12 April 2011

Abstract

Bioremediated soils are usually disposed of after meeting legislated guidelines defined by chemical and ecotoxicity tests. In many countries including Australia, ecotoxicity tests are not yet mandatory safety requirements. This study investigated the biotreatment of weathered hydrocarbon-contaminated soils in 12-week laboratory-based microcosms. Monitored natural attenuation resulted in ~43% reduction of total petroleum hydrocarbon contaminant to 5503 mg/kg (C16–C35), making the soil suitable for disposal as waste under current guidelines (pesticide and metal contents within safe limits). 16S rDNA (universal and AlkB) and ITS-based DGGE fingerprints showed stable and adapted microbial communities throughout the experimental period. However, ecotoxicology assays showed 100% mortality of earthworms (Eisena fetida) in potting soils containing ≥50% (≥2751 mg/kg, legally safe in situ concentrations) contaminated soil over 14 days. Up to 70% reduction in radish (Raphanus sativus) seed germination was observed in potting soils containing ≥10% contaminated soil (≥550 mg/kg, legally safe ex situ concentrations for soil disposal into residential areas). The results indicate the toxicity of these soils to soil biota despite meeting legislated Australian safe levels and guidelines for disposal or use in residential areas.

Additional keywords: bioremediation, DGGE, ecotoxicity test, ITS, 16S rDNA.


References

Adam G, Duncan H (2002) Influence of diesel fuel on seed germination. Environmental Pollution 120, 363–370.
Influence of diesel fuel on seed germination.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xos1WrtrY%3D&md5=78086c9010ab54c56f3eedec7438b2e3CAS | 12395850PubMed |

Anderson I, Campbell C, Prosser J (2003) Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. Environmental Microbiology 5, 36–47.
Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXhs1WntbY%3D&md5=21874dc9d0346a87742ade62273836b7CAS | 12542711PubMed |

Anderson I, Parkin P (2007) Detection of active soil fungi by RT-PCR amplification of precursor rRNA molecules. Journal of Microbiological Methods 68, 248–253.
Detection of active soil fungi by RT-PCR amplification of precursor rRNA molecules.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtVChurg%3D&md5=40d51fc74d35525056a00d8d1c75392dCAS | 17045683PubMed |

ANZECC/NHMRC (1992) ‘Australian and New Zealand Guidelines for the Assessment and Management of Contaminated Sites.’ (Australian and New Zealand Environmental and Conservation Council/National Health and Medical Research Council: Canberra, ACT)

ASTM (2004) ‘Standard guide for conducting laboratory soil toxicity or bioaccumulation tests with the Lumbricid earthworm Eisenia Fetida and the Enchytraeid potworm Enchytaeus albidus.’ ASTM Standard E 1676-04. pp. 201–219. (ASTM International: PA)

Banks M, Schwab P, Liu B, Kulakow P, Smith J, Kim R (2003) The effects of plants on the degradation and toxicity of petroleum contaminants in soil: A field investigation. In ‘Advances in biochemical engineering/biotechnology’. Vol. 78. pp. 75–96. (Springer: Berlin)

Bento FM, Camargo FAO, Okeke BC, Frankenberger WT (2005) Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation, and bioaugmentation. Bioresource Technology 96, 1049–1055.
Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation, and bioaugmentation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXnsFSjsg%3D%3D&md5=644ef6080edd5887181b81a84c0ac778CAS | 15668201PubMed |

Boonchan S, Britz M, Stanley G (2000) Degradation and minerlisation of high molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial co-cultures. Applied and Environmental Microbiology 66, 1007–1019.
Degradation and minerlisation of high molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial co-cultures.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXhsFyrt7g%3D&md5=42ecbb0f053bb4c8d8dd38d29a740c9cCAS | 10698765PubMed |

Dorn P, Salanitro J (2000) Temporal ecological assessment of oil contaminated soils before and after bioremediation. Chemosphere 40, 419–426.
Temporal ecological assessment of oil contaminated soils before and after bioremediation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXkt1OrtA%3D%3D&md5=1b0d414e604cef32c0335a7a2a5a62c6CAS | 10665408PubMed |

EPA (2010) Waste disposal information sheet. Current criteria for the classification of waste—including industrial and commercial waste (listed) and waste soil. Environmental Protection Authority, South Australia. Available at: www.epa.sa.gov.au/xstd_files/Waste/Information%20sheet/current_waste_criteria.pdf

Eriksson M, Dalhammar G, Borg-Karlson A (2000) Biological degradation of selected hydrocarbons in an old PAH/creosote contaminated soil from a gas work site. Applied and Environmental Microbiology 53, 619–626.

Evans FF, Rosado AS, Sebastián GV, Casella R, Machado PL, Holmström C, Kjelleberg S, van Elsas JD, Seldin L (2004) Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms. FEMS Microbiology Ecology 49, 295–305.
Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXlsl2hsro%3D&md5=ad2217d55113f22170066f5516fff0d8CAS | 19712422PubMed |

Genovese M, Denaro R, Cappello S, Marco GD, Spada GL, Giuliano L, Genovese L, Yakimov MM (2008) Bioremediation of benzene, toluene, ethylbenzene, xylenes-contaminated soil: a biopile pilot experiment. Journal of Applied Microbiology 105, 1694–1702.
Bioremediation of benzene, toluene, ethylbenzene, xylenes-contaminated soil: a biopile pilot experiment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsVyqs7fL&md5=2d3f03e74922692f66b18aae99fc4854CAS | 19149767PubMed |

Hamamura N, Fukui M, Ward D, Inskeep W (2008) Assessing soil microbial populations responding to crude-oil amendment at different temperatures using phylogenetic functional gene (alkB) and physiological analyses. Environmental Science & Technology 42, 7580–7586.
Assessing soil microbial populations responding to crude-oil amendment at different temperatures using phylogenetic functional gene (alkB) and physiological analyses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtV2lu7zE&md5=6e56e42bc617b3fa3e04c3e99bbec8bdCAS | 18983078PubMed |

Hubálek T, Vosahlova S, Mateju V, Kovacova N, Novotny C (2007) Ecotoxicity monitoring of hydrocarbon-contaminated soil during bioremediation: A case study, archives environmental contamination toxicology. Archives of Environmental Contamination and Toxicology 52, 1–7.
Ecotoxicity monitoring of hydrocarbon-contaminated soil during bioremediation: A case study, archives environmental contamination toxicology.Crossref | GoogleScholarGoogle Scholar | 17106791PubMed |

Husaini A, Roslin HA, Hii KSY, Ang CH (2008) Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites. World Journal of Microbiology & Biotechnology 24, 2789–2797.
Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht1OksLfE&md5=76b299c79aaebba3e9bb52fc24c2cc9bCAS |

ISO (2004) Soil quality. In ‘Determination of content of hydrocarbon in the range C10 to C40 by gas chromatography’. Vol. ISO 16703. (International Organisation for Standardisation: Geneva)

Kao C-M, Chen CS, Tsa F-Y, Yang K-H, Chien C-C, Liang S-H, Yang C-A, Chen SC (2010) Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume. Journal of Hazardous Materials 178, 409–416.
Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXksFSjsL8%3D&md5=083e5e61bc1950f9c22087f0a3a5c61fCAS | 20185233PubMed |

Lindley N (1992) Hydrocarbon-degrading yeasts and filamentous fungi of biotechnology importance. In ‘Handbook of applied mycology’. Vol. 4. (Eds D Arora, R Elander, K Mukerji) pp. 905–930. (Dekker: New York)

Makadia TH, Adetutu EM, Simons KL, Jardine D, Sheppard PJ, Ball AS (2011) Re-use of remediated soils for the bioremediation of waste oil sludge. Journal of Environmental Management 92, 866–871.

Manceralopez M, Garcia F, Gomez B, Vazquez R, Castanda G, Cortes J (2008) Bioremediation of an aged hydrocarbon-contaminated soil by a combined system of biostimulation-bioaugmentation with filamentous fungi. International Biodeterioration & Biodegradation 61, 151–160.
Bioremediation of an aged hydrocarbon-contaminated soil by a combined system of biostimulation-bioaugmentation with filamentous fungi.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhslOltLc%3D&md5=54a08cee847d8d9acf83459007b218e5CAS |

Mao D, Lookman R, Van De Weghe H, Weltens R, Vanerman G, De Brucker N, Diels L (2009) Estimation of ecotoxicity of petroleum hydrocarbon mixtures in soil based on HPLC–GCXGC analysis. Chemosphere 77, 1508–1513.
Estimation of ecotoxicity of petroleum hydrocarbon mixtures in soil based on HPLC–GCXGC analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsVajtLnP&md5=ee40d30329de8d564d47146ad733d9c5CAS | 19879629PubMed |

Margesin R, Hammerle M, Tscherko D (2007) Microbial activity and community composition during bioremediation of diesel-oil-contaminated soil: effects of hydrocarbon concentration, fertilizers, and incubation time. Microbial Ecology 53, 259–269.
Microbial activity and community composition during bioremediation of diesel-oil-contaminated soil: effects of hydrocarbon concentration, fertilizers, and incubation time.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhslCltb0%3D&md5=96e94ca9cb6bd19372ffa8f681f7d642CAS | 17265002PubMed |

Martin-Gill J, Navas-Gracias L, Gomez-Sobrino E, Correa-Guimaraes A, Hernandez-Navarro S, Sanchez-Bascones M, Ramos-Sanchez M (2008) Composting and vermicomposting experiences in the treatment and bioconversion of asphaltenes from the Prestige oil spill. Bioresource Technology 99, 1821–1829.
Composting and vermicomposting experiences in the treatment and bioconversion of asphaltenes from the Prestige oil spill.Crossref | GoogleScholarGoogle Scholar | 17512195PubMed |

McCaig A, Glover L, Prosser J (2001) Numerical analysis of grassland bacterial community structure under different land management regimens by using 16S ribosomal DNA sequence data and denaturing gradient gel electrophoresis banding patterns. Applied and Environmental Microbiology 67, 4554–4559.
Numerical analysis of grassland bacterial community structure under different land management regimens by using 16S ribosomal DNA sequence data and denaturing gradient gel electrophoresis banding patterns.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXns1WisLs%3D&md5=6cc82891556d1f1d3a7654e158143d31CAS | 11571155PubMed |

Mishra S, Jyot J, Kuhad R, Lal B (2001) Evaluation of inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil. Applied and Environmental Microbiology 67, 1675–1681.
Evaluation of inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXis1egt7w%3D&md5=ac7707825c4d262ec91c0e3940f31efdCAS | 11282620PubMed |

Molina-Barahona L, Vega-Loyo L, Guerrero M, Ramirez S, Romero I, Vega-Jurguin C, Albores A (2005) Ecotoxicity evaluation of diesel-contaminated soil before and after a bioremediation process. Environmental Toxicology 20, 100–109.
Ecotoxicity evaluation of diesel-contaminated soil before and after a bioremediation process.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhvVeqtrk%3D&md5=9faa740d71cab85d593880288e4d71afCAS | 15712321PubMed |

Muyzer G, Waal ED, Uitterlinden A (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695–700.

NEPC (1999) Assessment of site contamination. Schedule B(1) Guideline on the investigation levels for soil and groundwater. National Environmental Protection Council. Available at: www.ephc.gov.au/sites/default/files/ASC_NEPMsch__01_Investigation_Levels_199912.pdf (accessed 01/10/2010)

Parrish Z, White J, Isleyen M, Gent M, Iannucci-Berger W, Eitzer B, Kelsey J, Mattina M (2006) Accumulation of weathered polycyclic aromatic hydrocarbons (PAHs) by plant and earthworm species. Chemosphere 64, 609–618.
Accumulation of weathered polycyclic aromatic hydrocarbons (PAHs) by plant and earthworm species.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xmt1Okt7w%3D&md5=bf8c941c2aadb5c3e346ae352dc949c0CAS | 16337258PubMed |

Peng S, Zhou Q, Cai Z, Zhang Z (2009) Phytoremediation of petroleum contaminated soils by Mirabilis jalapa L. in a greenhouse plot experiment. Journal of Hazardous Materials 168, 1490–1496.
Phytoremediation of petroleum contaminated soils by Mirabilis jalapa L. in a greenhouse plot experiment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXnslKiu7k%3D&md5=61993d121a34e477b6a9ac88ae45ddcbCAS | 19346069PubMed |

Philip JC, Bamforth SM, Singleton I, Atlas RM (2005) Environmental pollution and restoration: a role for bioremediation. In ‘Bioremediation: applied microbial solutions for real world environmental clean up’. (Eds RM Atlas, JC Philip) pp. 1–48. (ASM Press: Washington, DC)

Plaza G, Nalecz-Jawecki G, Ulfig K, Brigmon R (2005) The application of bioassays as indicators of petroleum-contaminated soil remediation. Chemosphere 59, 289–296.
The application of bioassays as indicators of petroleum-contaminated soil remediation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhslWmsr8%3D&md5=6efd98f7221e124707258fd91291a85fCAS | 15722101PubMed |

Rojo F (2009) Degradation of alkanes by bacteria. Environmental Microbiology 11, 2477–2490.
Degradation of alkanes by bacteria.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlKisLzP&md5=845053bd0f1860d0f0735b8d5094692dCAS | 19807712PubMed |

Salanitro J, Dorn P, Huesemann M, Moore K, Rhodes I (1997) Crude oil hydrocarbon bioremediation and soil ecotoxicity assessment. Environmental Science & Technology 31, 1769–1776.
Crude oil hydrocarbon bioremediation and soil ecotoxicity assessment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXislSltLw%3D&md5=b72e2c2134520eb34ffe0dc215b910b4CAS |

Schultz E, Joutti A, Raisanen M-L, Lintinen P, Martikainen E, Lehto O (2004) Extractability of metals and ecotoxicity of soils from two old wood impregnation sites in Finland. The Science of the Total Environment 326, 71–84.
Extractability of metals and ecotoxicity of soils from two old wood impregnation sites in Finland.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXktVChs7k%3D&md5=318bc572b2c482a35596467b3645e4f0CAS | 15142767PubMed |

Vinas M, Sabate J, Espuny M, Solanas A (2005) Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creseote-contaminated soil. Applied and Environmental Microbiology 71, 7008–7018.
Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creseote-contaminated soil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXht1ekur3L&md5=fa32df8a3331d6bbdb803cc186ba34feCAS | 16269736PubMed |

Wang S, Yan Z, Guo G, Lu G, Wang Q, Li F (2010) Ecotoxicity assessment of aged petroleum sludge using a suite of effects-based end points in earthworm Eisenia fetida. Environmental Monitoring and Assessment 169, 417–428.
Ecotoxicity assessment of aged petroleum sludge using a suite of effects-based end points in earthworm Eisenia fetida.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtFSnt7rM&md5=10b53782e5d3cec9e53fd567daa43830CAS | 19844801PubMed |

Wentzel A, Ellingsen T, Kotlar H-K, Zotchev S, Throne-Holst M (2007) Bacterial metabolism of long-chain n-alkanes. Applied Microbiology and Biotechnology 76, 1209–1221.
Bacterial metabolism of long-chain n-alkanes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtVGmt7%2FN&md5=75e96ea5ee977aff03ef883003b55158CAS | 17673997PubMed |

Wu Y, Luo Y, Zou D, Ni J, Lui W, Teng Y, Li Z (2008) Bioremediation of polycyclic aromatic hydrocarbons contaminated soil with Monilinia sp., degradation and microbial community analysis. Biodegradation 19, 247–257.
Bioremediation of polycyclic aromatic hydrocarbons contaminated soil with Monilinia sp., degradation and microbial community analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXitl2jtL0%3D&md5=cd021dd2ca07b1d1aa922c4d5e6103f2CAS | 17541708PubMed |