Volume 8, Issue 10 p. 1736-1745

Identification and quantification of uncultivated Proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil

David R. Singleton

David R. Singleton

Department of Environmental Sciences and Engineering School of Public Health, CB #7431, University of North Carolina, Chapel Hill, NC 27599-7431, USA.

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Ramiah Sangaiah

Ramiah Sangaiah

Deceased.

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Avram Gold

Avram Gold

Department of Environmental Sciences and Engineering School of Public Health, CB #7431, University of North Carolina, Chapel Hill, NC 27599-7431, USA.

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Louise M. Ball

Louise M. Ball

Department of Environmental Sciences and Engineering School of Public Health, CB #7431, University of North Carolina, Chapel Hill, NC 27599-7431, USA.

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Michael D. Aitken

Corresponding Author

Michael D. Aitken

*E-mail [email protected]; Tel. (+1) 919 966 1481; Fax (+1) 919 966 7911. Search for more papers by this author
First published: 15 August 2006
Citations: 107

Summary

Uncultivated bacteria associated with the degradation of pyrene in a bioreactor treating soil contaminated with polycyclic aromatic hydrocarbons (PAH) were identified by DNA-based stable-isotope probing (SIP) and quantified by real-time quantitative PCR. Most of the 16S rRNA gene sequences recovered from 13C-enriched DNA fractions clustered phylogenetically within three separate groups of β- and γ-Proteobacteria unassociated with described genera and were designated ‘Pyrene Groups 1, 2 and 3’. One recovered sequence was associated with the Sphingomonas genus. Pyrene Groups 1 and 3 were present in very low numbers in the bioreactor but represented 75% and 7%, respectively, of the sequences recovered from 16S rRNA gene clone libraries constructed from 13C-enriched DNA. In a parallel time-course incubation with unlabelled pyrene, there was between a 2- and 4-order-of-magnitude increase in the abundance of 16S rRNA genes from Pyrene groups 1 and 3 and from targeted Sphingomonas spp. over a 10 day incubation. Sequences from Pyrene Group 2 were 11% of the SIP clone libraries but accounted for 14% of the total bacterial 16S rRNA genes in the bioreactor community. However, the abundance of this group did not increase significantly in response to pyrene disappearance. These data indicate that the primary pyrene degraders in the bioreactor were uncultivated, low-abundance β- and γ-Proteobacteria not previously associated with pyrene degradation.