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Applied and Environmental Microbiology, February 2008, p. 1176-1182, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01518-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Enrichment, Isolation, and Phylogenetic Identification of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria from Elizabeth River Sediments ^{, ,}

Edward J. Hilyard,^1, Joanne M. Jones-Meehan,² Barry J. Spargo,¹ and Russell T. Hill^3*

Naval Research Laboratory, Washington, DC 20375,¹ Department of Homeland Security, Washington, DC 20528,² Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202³

Received 5 July 2007/ Accepted 12 December 2007

The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of ¹⁴C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.

Published ahead of print on 21 December 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Contribution no. 07-164 from the Center of Marine Biotechnology.

Present address: Naval Medical Research Center, Silver Spring, MD 20910.