Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10 2 to 10 3 gene copies, which was lowered to 10 0 to 10 1 gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivationbased molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

Although aromatic compounds are most often present in the environment as components of complex mixtures, biodegradation studies commonly focus on the degradation of individual compounds. The present study was performed to investigate the range of aromatic substrates utilized by biphenyl- and naphthalene-degrading environmental isolates and to ascertain the effects of co-occurring substrates during the degradation of mono-aromatic compounds. Bacterial strains were isolated on the basis of their ability to utilize either biphenyl or naphthalene as a sole source of carbon. Growth and transformation assays were conducted on each isolate to determine the range of substrates degraded. One isolate, Pseudomonas putida BP18, was tested for the ability to biodegrade benzene, toluene, ethylbenzene and xylene isomers (BTEX) individually and as components of mixtures. Overall, the results indicate that organisms capable of growth on multi-ring aromatic compounds may be particularly versatile in terms of aromatic hydrocarbon biodegradation. Furthermore, growth and transformation assays performed with strain BP18 suggest that the biodegradation of BTEX and biphenyl by this strain is linked to a catabolic pathway with overlapping specificities. The broad substrate specificity of these environmental isolates has important implications for bioremediation efforts in the field.

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were to increase utilization of bioremediation by INDOT and other agencies for the remediation of petroleum contaminated sites, reduce uncertainty associated with the design and implementation of bioremediation systems, reduce reliance on landfills for disposal of contaminated soils, reduce long-term liability associated witii hazardous v,aste. and to improve the quality of engineering science utilized for the design of bioremediation systems. In addition, a guidance manual for remediation decision makers was developed. The benefits of achieving these objectives will be to decrease costs associated with Leaking Underground Storage Tank (LUST) remediation, improve environmental qualit>-, and to improve pubhc and environmental health. To achieve the objectives outiined above several bioremediation field demonstrations were successfully developed. A bioremediation field demonstration using Monitored Natural .•\ttenuation was implemented and is an ongoing project at Linton, Indiana. A land farming field demonstration using excavated low hydraulic conducti%-ity soils has been completed at Chrisney, Indiana. An engineered bioremediation field demonstration at a site with contaminated groundwater using a combined Air Sparging-Soil Vapor Extraction system is underway at Shoals, Indiana.17.