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Functional genes reveal the intrinsic PAH biodegradation potential in creosote-contaminated groundwater following in situ biostimulation

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Abstract

A small-scale functional gene array containing 15 functional gene probes targeting aliphatic and aromatic hydrocarbon biodegradation pathways was used to investigate the effect of a pilot-scale air sparging and nutrient infiltration treatment on hydrocarbon biodegradation in creosote-contaminated groundwater. Genes involved in the different phases of polycyclic aromatic hydrocarbon (PAH) biodegradation were detected with the functional gene array in the contaminant plume, thus indicating the presence of intrinsic biodegradation potential. However, the low aerobic fluorescein diacetate hydrolysis, the polymerase chain reaction (PCR) amplification of 16S rRNA genes closely similar to sulphate-reducing and denitrifying bacteria and the negligible decrease in contaminant concentrations showed that aerobic PAH biodegradation was limited in the anoxic groundwater. Increased abundance of PAH biodegradation genes was detected by functional gene array in the monitoring well located at the rear end of the biostimulated area, which indicated that air sparging and nutrient infiltration enhanced the intrinsic, aerobic PAH biodegradation. Furthermore, ten times higher naphthalene dioxygenase gene copy numbers were detected by real-time PCR in the biostimulated area, which was in good agreement with the functional gene array data. As a result, functional gene array analysis was demonstrated to provide a potential tool for evaluating the efficiency of the bioremediation treatment for enhancing hydrocarbon biodegradation in field-scale applications.

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Acknowledgements

This work was supported by the Ekokem Environmental Foundation (Finland). We thank Dr. Kimmo Järvinen from Ramboll Finland Ltd. for site introduction and Dr. Ari Laitinen from Doranova Ltd. for providing technical information on the biostimulation treatment. We also thank the Finnish DNA Microarray Centre for the preparation of the functional gene arrays. Dr. Wolfgang Hillen is thanked for supplying the A. calcoaceticus strain ADP1, Dr. Tim Tolker-Nielsen for providing the fluorescently tagged P. putida strain OUS82, and Dr. Timo Pulli for the morphine-specific Fab fragment cDNA. Marjo Öster we thank for her assistance in the PCR amplification of the dsrA genes.

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Authors and Affiliations

  1. Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland

    Mari Nyyssönen, Anu Kapanen & Merja Itävaara

  2. WSP Environmental Ltd., Heikkiläntie 7, 00210, Helsinki, Finland

    Reetta Piskonen

  3. Ramboll Finland Ltd., Porrassalmenkatu 1, 50100, Mikkeli, Finland

    Tuomas Lukkari

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  1. Mari Nyyssönen
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Correspondence to Mari Nyyssönen.

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Table S1

PCR primers and thermal profiles used for the amplification of the gene probes designed in this study (PDF 8 kb)

Fig. S2

Average PAH concentrations in the creosote-contaminated groundwater during treatment by air sparging and nutrient infiltration. The groundwater samples were recovered from monitoring wells BST1 and BST2 located upstream from the biostimulation treatment zone and monitoring wells BST3, BST4 and BST5 located downstream from it (Fig. 1b). The error bars represent standard deviations between three monitoring wells. a PAH (EPA 16), b naphthalene, c fluorene and d phenanthrene (PDF 7 kb)

Table S3

Specificity of gene probes attached on the functional gene array determined by hybridisation with pure bacterial cultures. The average signal intensities and standard deviations were determined from unnormalised hybridisation signals measured from six replicate spots. The standard deviations between replicate spots are presented in parenthesis (PDF 6 kb)

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Nyyssönen, M., Kapanen, A., Piskonen, R. et al. Functional genes reveal the intrinsic PAH biodegradation potential in creosote-contaminated groundwater following in situ biostimulation. Appl Microbiol Biotechnol 84, 169–182 (2009). https://doi.org/10.1007/s00253-009-2022-8

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