3.1 Sampling
Sediment samples were collected from Chattanooga Creek, a shallow, lotic system in October 2004, and April, July, and October 2005. Samples were collected from a site upstream of the Superfund Site (W), from a section that was excavated in 1998 (DO), and from an unremediated, highly contaminated site (CF) (Figure II-1) (corresponding to sites 1, 4, and 6, respectively, from Dionisi et al. 2004). The control site (W) is upstream of the highly contaminated area, however still exhibits low levels of contamination due to heavy industry and traffic in the area. At all sites, surface sediments were fine sand, with no visible coal tar (unlike deeper sediments). Triplicate sediment samples were taken from the top 5-10 (aerobic zone), stored in Whirlpak bags, and either frozen
immediately on dry ice for DNA extraction or kept at ambient temperature for mineralization assays.
3.2 PAH quantification
PAHs were extracted according to EPA method 3546 using the Microwave Automated Reaction System from CEM (Matthews, NC). Briefly, 10 g sediment were combined with Base/Neutral Surrogate Standard (UltraScientific, North Kingstown, RI) and 5 g Na2SO4. Samples were ground to < 1 mm particle sizes, extracted twice in GreenChem vessels with 25 ml of a 1:1 hexanes:acetone mixture (HPLC grade, Fisher Scientific, Pittsburg, PA) according to manufacturer’s protocol (100°C for at least 20 minutes). The solvent was concentrated and run on Hewlett-Packard gaschromatograph (model 6890) equipped with a mass-selective detector(model 5973) and Hewlett-Packard
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Figure II-1. Map of sampling locations on Chattanooga Creek.
The Tennessee River is pictured in the top left hand corner; the Tennessee-Geoergia border is along the bottom. Zone I is upstream of point sources of contamination (sources include the Tennessee products coking coal carbonization facility, in operation from 1918-1987). Zone II sediments were excavated in 1998; Zone III is currently being excavated (2006-2007).
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autosampler complying with EnvironmentalProtection Agency (EPA) standard method 8270D. Sediment dry weight was determined following baking of 5 g of wet soil at >80°C for at least 48 hours.
3.3 Mineralization Assays
To determine mineralization rates, three vials (and one killed control) were used for each sample containing 2 g sediment slurried with 1 ml dH2O in a 40 ml EPA vial with a septum. As a positive control, a 1 ml overnight culture of
Mycobacterium flavescens (ATCC 700033) (pyrene) or Pseudomonas fluorescens HK44 (naphthalene) were used in place of sediment. One ml 2N H2SO4 was added to each killed control; an 8 ml vial with 0.5 ml 0.5N NaOH was placed in each to serve as a CO2 trap. Either 400,000 dpm naphthalene-1-14C (31.3 mCi/mmol, Sigma, St. Louis, MO) or 200,000 dpm pyrene-4,5,9,10-14C (55 mCi/mmol, Sigma, St. Louis, MO) dissolved in acetone were added to each.
Vials were dark-incubated at room temperature with shaking for 40 hours. The NaOH was mixed with water and ReadySafe liquid scintillation cocktail
(Beckman Coulter, Fullerton, CA) and assayed on a Packard TriCarb 2900TR Liquid Scintillation Analyzer (PerkinElmer, Downers Grove, IL). 14C efficiencies (0-156 keV) ranged from 94.37 to 96.18.
3.4 DNA Extraction and Real-time PCR
Total DNA was purified in triplicate from sediment samples usingthe FastDNA SPIN kit for soil (Qbiogene, Morgan Irvine, CA) with minor modifications as described previously (Dionisi et al. 2004). Primer and probe sets for quantifying dioxygenase genes targeted genes encoding the large (α) dioxygenase subunit (nidA, nagAc, and nahAc). For the nidA TaqMan® probe quantitative PCR assay,
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primers and probes target conserved regions determined from a multiple alignment of nidA from several PAH-degrading Mycobacterium: M. flavescens (AF548343), M. fredrickbergense (AF548345), M. gilvum (AF548347), and M.
sp. strains PYR-1 (AF249301), 6PYR (AJ494745), JLS (AY330098), KMS (AY330100), MCS (AY33010), and MHP-1 (AB179737). The TaqMan® probe (5’-FAM-TCCTACCCGTCGCCGGTACA-BHQ1) contained several base pair differences to closely related Rhodococcus sp. NCIMB narA (AF082663) gene.
Forward and reverse primer sequences were
5’-TTCCCGAGTACGAGGGATAC, and 5’-TCACGTTGATGAACGACAAA, respectively. The real-time PCR reactions used QuantiTect Probe PCR Master Mix (Qiagen, Valencia, CA) and were performed on an MJ Opticon thermocycler (Bio-Rad, Hercules, CA) using the following protocol: 50°C for 2 minutes, 95°C for 15 minutes, then 40 cycles of denaturing at 94°C for 15 seconds and annealing at 56°C for 30 seconds. nidA from Mycobacterium flavescens (ATCC 700033) cloned into TOPO-TA-PCR4.1 (Invitrogen, Carlsbad, CA) vector was used to create an internal eight-point standard curve ranging from 101 to 108 gene copies per reaction. To ensure specificity of the assay, PCR products were cloned.
Twenty clones were sequenced using M13 reverse primer on an ABI3100 genetic analyzer (Applied Biosystems, Foster City, CA) at the Molecular Biology
Resource Facility, University of Tennessee (Knoxville, TN).
A TaqMan® probe quantitative PCR assay targeting conserved regions of nahAc genes encoding naphthalene dioxygenase in γ-proteobacteria was also developed.
Eighteen Pseudomonas were used to design degenerate primers and probe, including, P. stutzeri (AF306425), P. balearica (AF306428), P. fluorescens (AY125981), P. putida strains (AF306430, AF306441, AF306439), and related Pseudomonas sp. Forward and reverse primer sequences were
5’-CAGAGCGTYCCRTTYGAAAA and 5’-TCGAAGCAACCRTARATGAA, respectively. The TaqMan® probe sequence was
5’-FAM-TGGGGTTGAAAGAAGTCGCTCG-BHQ1. Real-time PCR assays were
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performed as described above, using an annealing temperature of 52°C, and cloned nahA from Pseudomonas putida G7 as the internal standard. Copies of nagAc-like genes in β-proteobacteria as well as universal 16S rDNA genes were quantified as previously described (Harms et al. 2003, Dionisi et al. 2004).
Statistical comparisons of gene abundance were done in NCSS (Number Cruncher Statistical Systems, Kaysville, UT) using a nonparametric one-way ANOVA on the ranked data, and Kruskal-Wallis Multiple Comparison Z-test with Bonferroni adjustment. Comparisons were made between sites (W, DO, CF) and between sampling dates; ANOVA results with p < 0.01 were considered statistically significant.
3.5 Mycobacterium 16S clone library
Primers designed by Leys et al. (Leys et al. 2005b) to target an approximately 530 bp region of the 16S ribosomal gene in fast-growing Mycobacterium
(corresponding to positions 66 and 600 in Escherichia coli) were used to create a clone library using DNA extracted from site DO. A touchdown PCR protocol was used (95°C for 10 minutes, then 20 cycles of denaturing at 95°C for 30s, annealing at 55°C - 1°C/cycle for 30s and extension 72°C for 30s, followed by 10 cycles with an annealing step at 45°C, and a final extension of 72°C for 10
minutes). PCR products were cloned into a TOPO-TA-PCR4.1 vector (Invitrogen, Carlsbad, CA). Sequencing was done as described above.
Alignments and phylogenetic trees were done using MEGA (Molecular Evolutionary Genetics Analysis) software version 3.1
(http://www.megasoftware.net/). Sequences were deposited in the NCBI GenBank, accession numbers EF438310-EF438383.
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