Microbial Community Structure and Biodegradation Activity of Particle-Associated Bacteria in a Coal Tar Contaminated Creek JENNIFER M. DEBRUYN † AND GARY S. SAYLER* ,†,‡ Center for Environmental Biotechnology and Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996 Received November 27, 2008. Revised manuscript received February 18, 2009. Accepted February 23, 2009. The Chattanooga Creek Superfund site (Chattanooga, TN) is one of the most polluted waterways in the southeastern U.S. with high polycyclic aromatic hydrocarbon (PAH) concentrations in the sediments. PAHs associate with suspended solids in the water column, and may be redeposited onto the floodplain. These suspended particles represent an interesting but understudied environment for PAH-degrading microbial communities. This study tested the hypotheses that particle- associated bacterial (PAB) communities have genotypic potential (PAH-dioxygenase genes) and activity (naphthalene and pyrene mineralization), and can contribute to natural attenuation of PAHs in Chattanooga Creek. Upstream of the Superfund site, mineralization ranged from 0.2 to 2.0% of added 14 C- naphthalene and 0 to 0.1% 14 C-pyrene (after 40 h), with first order biodegradation rate constants ( k 1 ) ranging from 1.09 to 9.18 × 10 -5 h -1 and 0 to 1.13 × 10 -6 h -1 , respectively. Mineralization was significantly greater in PAB communities within the contaminated zone, with 11.8 to 31.2% 14 C-naphthalene ( k 1 5.34 to 14.2 × 10 -4 h -1 ) and 1.3 to 6.6% 14 C-pyrene mineralized ( k 1 2.89 to 15.0 × 10 -5 h -1 ). Abundances of nagAc (naphthalene dioxygenase) and nidA (pyrene dioxygenase) genes indicated that PAB communities harbored populations with genetic potential for both low- and high-molecular weight PAH degradation, and quantification of Mycobacterium 16S rDNA genes indicated that PAH-degrading mycobacteria are also prevalent in this environment. Phylogenetic comparisons (T-RFLPs) between PAB and sediments indicated these microbial communities were taxonomically distinct, but shared some functional similarities, namely PAH catabolic genotypes, mineralization capabilities, and community structuring along a contamination gradient. Introduction In an aquatic system, hydrophobic contaminants like poly- cyclic aromatic hydrocarbons (PAHs) partition into sedi- ments, where microbial biodegradation is well-documented and generally the primary route of attenuation of these carcinogenic and mutagenic pollutants (1). In addition to lake- or stream-bottom sediments, PAHs will also associate with particles in the water column, either through diffusion and subsequent sorption onto suspended particles, or through disturbance and suspension of contaminated stream- bottom sediments (2-4). The Chattanooga Creek Superfund Site, located in an industrial and residential area of Chattanooga, Tennessee, was placed on the National Priorities list in 1995 due to mixed priority pollutants and heavy coal tar contamination pre- dominantly from a nearby coking coal plant (5). This has resulted in high concentrations of PAHs in the sediments, despite physical remediation efforts (6, 7). In addition, repetitive deposition of PAHs onto floodplain soils has been documented (8). Flood events can redistribute organic contaminants (9), and frequent flooding of Chattanooga Creek is likely contributing to observed PAHs on the floodplains. Particles suspended in the water column act as a vehicle for contaminant transport both within an aquatic system (2) and onto floodplains. This leads to the hypothesis that microbial communities associated with these particles represent one pathway of PAH attenuation. Higher densities of bacteria on particles may increase contact with PAHs and may aid bacteria in accessing insoluble PAHs (10). In addition, suspension in the water column, especially in a lotic creek such as Chattanooga Creek, results in an oxygenated environment conducive to aerobic biodegradation. There is evidence that biodegradation of PAHs increases with in- creasing suspended sediment loads (11), indicating that particle-associated bacteria (PAB) are likely contributing to PAH attenuation in aquatic systems. Biodegradative potential, through quantification of both low-and high- molecular weight PAH dioxygenase genes (nidA, nagAc, and nahAc), as well as mineralization activity have already been documented for sediment communities in Chattanooga Creek (6, 7). In particular, detection of nidA in combination with a phylo- genetic clone library from the sediments highlighted popu- lations of fast-growing Mycobacterium spp. (7) recently characterized for their ability to degrade high molecular weight PAHs (12). Suspended particles, also referred to as aggregates or flocs, have been recognized as a unique environment within the aquatic system, comprised of both inorganic (e.g., clay) and organic (e.g., detritus) materials, supporting PAB communi- ties (13, 14). These PAB are important to carbon metabolism and cycling in a variety of aquatic environments (15). In both marine and freshwater environments, PAB communities tend to have higher densities (14, 16), higher metabolic and enzymatic activities (17, 18), and are phylogenetically distinct compared to free-living bacteria (19-21). While other studies have contrasted free-living bacteria and PAB with an emphasis on phylogenetic relationships, this study compared PAB with sediment communities emphasizing functional (biodegradative) differences. Sus- pended particle systems are very dynamic, however little baseline information exists regarding molecular biodegra- dative potential of PAB communities. Along with quantifica- tion of PAH degradative genes, this study also aimed to determine prevalence of particle-associated PAH-degrading mycobacteria. Specifically, this study addressed the hypoth- eses that PAB communities are unique in structure (phy- logeny) and function (low- and high-molecular-weight PAH biodegradation) compared to the sediment communities, and contribute to natural attenuation of PAHs in Chattanooga Creek. * Corresponding author phone: 865-974-8080; fax: 865-974-8086; e-mail: sayler@utk.edu. † Center for Environmental Biotechnology. ‡ Department of Microbiology. Environ. Sci. Technol. 2009, 43, 3047–3053 10.1021/es803373y CCC: $40.75  2009 American Chemical Society VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3047 Published on Web 03/27/2009